Antibody against the csf-1r

ABSTRACT

The present invention provides antibodies specific for the CSF-1R, compositions comprising said antibodies and methods of treatment using such compositions.

The CSF-1 (Colony-Stimulating Factor-1) is a cytokine expressedparticularly by various types of cells. It is a differentiation, growthand survival factor for cells of the mononuclear phagocyte lineage whichexpress the receptor for CSF-1 (CSF-1R) (SHERR. Colony-stimulatingfactor-1 receptor. blood. 1990, vol. 75, no. 1, p. 1-12.). CSF-1R is atyrosine kinase receptor encoded by the c-fms protoonogene containing anintracellular kinase domain and a ligand-binding extracellular regionorganized in five immunoglobulin-like subdomains. Response to CSF-1results in increased survival, growth, differentiation, and reversiblechanges in function. The c-fms gene is itself a macrophagedifferentiation marker. The extent of c-fms expression is stronger thanthat of other macrophage-specific genes including lysozyme and amacrophage-specific protein tyrosine phosphatase. (HUME, et al.Regulation of CSF-1 receptor expression. Molecular reproduction anddevelopment. 1997, vol. 46, no. 1, p. 46-52.).

In addition to cells of the mononuclear phagocyte lineage, the CSF-1R isalso expressed by many types of human tumors. In breast cancer, CSF-1Rexpression is associated with larger tumor sizes and decreased survival(KLUGER, et al. Macrophage colony-stimulating factor-1 receptorexpression is associated with poor outcome in breast cancer by largecohort tissue microarray analysis. Clinical cancer research. 2004, vol.10, no. 1, p. 173-7.; SCHOLL, et al. Anti-colony-stimulating factor-1antibody staining in primary breast adenocarcinomas correlates withmarked inflammatory cell infiltrates and prognosis. Journal of theNational Cancer Institute. 1994, vol. 86, no. 2, p. 120-6.). Inepithelial ovarian cancer, the majority of primary tumors and metastasesstrongly express the CSF-1R, and metastases frequently co-express CSF-1and CSF-1R. The CSF-1R is also expressed by tumor-infiltratingmacrophages (CHAMBERS, et al. Overexpression of epithelial macrophagecolony-stimulating factor (CSF-1) and CSF-1 receptor: a poor prognosticfactor in epithelial ovarian cancer, contrasted with a protective effectof stromal CSF-1. Clinical Cancer Research. 1997, vol. 3, no. 6, p.999-1007.). In ovarian and endometrial cancers, Northern blot analysisshows that the vast majority of tumors co-express CSF-1 and CSF-1R,while CSF-1R expression is only weakly detected in normal endometrialtissue samples (BAÏOCCHI, et al. Expression of the macrophagecolony-stimulating factor and its receptor in gynecologic malignancies.Cancer. 1991, vol. 67, no. 4, p. 990-6.). In cervical carcinomas, CSF-1Rexpression is up-regulated both in tumor stroma and in tumor epithelium,compared with normal endometrium (KIRMA, et al. Elevated expression ofthe oncogene c-fms and its ligand, the macrophage colony-stimulatingfactor-1, in cervical cancer and the role of transforming growthfactor-beta1 in inducing c-fms expression. Cancer res. 2007, vol. 67,no. 5, p. 1918-26.). In renal carcinoma, infiltration oftumor-associated macrophages expressing high levels of CSF-1R isassociated with tumor progression (HEMMERLEIN, et al. Expression ofacute and late-stage inflammatory antigens, c-fms, CSF-1, and humanmonocytic serine esterase 1, in tumor-associated macrophages of renalcell carcinomas. Cancer immunology, immunotherapy 2000, vol. 49, no. 9,p. 485-92.). CSF-1R is expressed by close to 100% prostaticintraepithelial neoplasia or cancer samples (IDE, et al. Expression ofcolony-stimulating factor 1 receptor during prostate development andprostate cancer progression. Proc. Natl. Acad. Sci U.S.A. 2002, vol. 99,no. 22, p. 14404-9.). CSF-1R expression has also been detected in acutemyeloblastic leukemias and B-cell chronic lymphocytic leukemias(RAMBALDI, et al. Expression of the macrophage colony-stimulating factorand c-fms genes in human acute myeloblastic leukemia cells. Journal ofClinical Investigation. 1988, vol. 81, no. 4, p. 1030-5.).

Work done by immunohistochemistry and in situ hybridization hasdemonstrated specificity of the expression of CSF-1 in invasive breastcancer cells while such production is not observed in intra-canal ornon-invasive tumor cells (SCHOLL, et al. Anti-colony-stimulatingfactor-1 antibody staining in primary breast adenocarcinomas correlateswith marked inflammatory cell infiltrates and prognosis. Journal of theNational Cancer Institute. 1994, vol. 86, no. 2, p. 120-6.; TANG, et al.M-CSF (monocyte colony stimulating factor) and M-CSF receptor expressionby breast tumour cells: M-CSF mediated recruitment of tumourinfiltrating monocytes?. Journal of cellular biochemistry. 1992, vol.50, no. 4, p. 350-6.). Production of CSF-1 by invasive tumor cellscorrelates with its increase in concentration in the plasma of patients,where it can exceed 1000 pg/ml compared with less than 300 pg/ml innormal subjects. High serum concentration correlates with advancedstages of the disease and unfavorable short term prognostic (SCHOLL, etal. Circulating levels of colony-stimulating factor 1 as a prognosticindicator in 82 patients with epithelial ovarian cancer. British journalof cancer. 1994, vol. 69, no. 2, p. 342-6.; SCHOLL. Circulating levelsof the macrophage colony stimulating factor CSF-1 in primary andmetastatic breast cancer patients. A pilot study. Breast cancer researchand treatment. 1996, vol. 39, no. 3, p. 275-83.). Moreover, it has beendemonstrated that CSF-1 stimulates mobility and invasiveness of tumorcells (DORSCH, et al. Macrophage colony-stimulating factor gene transferinto tumor cells induces macrophage infiltration but not tumorsuppression. European journal of immunology. 1993, vol. 23, no. 1, p.186-90.; WANG, et al. Induction of monocyte migration by recombinantmacrophage colony-stimulating factor. Journal of immunology. 1988, vol.141, no. 2, p. 575-9.; FILDERMAN, et al. Macrophage colony-stimulatingfactor (CSF-1) enhances invasiveness in CSF-1 receptor-positivecarcinoma cell lines. Cancer res. 1992, vol. 52, no. 13, p. 3661-6.).

CSF-1 also has a chemotactic effect on precursors of the myeloid line,which facilitates infiltration of monocytes in the tumor. However, thepresence of these monocytes is not sufficient to observe destruction ofthe tumor by the immune system. (DORSCH, et al. Macrophagecolony-stimulating factor gene transfer into tumor cells inducesmacrophage infiltration but not tumor suppression. European journal ofimmunology. 1993, vol. 23, no. 1, p. 186-90.). It appears that at thehigh serum contents commonly found in patients suffering from tumors ofthe breast, ovaries or pancreas, CSF-1 orients the differentiation ofthese monocytes into macrophages and not into dendritic cells capable ofpresenting tumoral antigens and thus initiating an efficient cytotoxicimmune response directed against tumor cells (SCHOLL. Circulating levelsof the macrophage colony stimulating factor CSF-1 in primary andmetastatic breast cancer patients. A pilot study. Breast cancer researchand treatment. 1996, vol. 39, no. 3, p. 275-83.; BARON, et al.Modulation of MHC class II transport and lysosome distribution bymacrophage-colony stimulating factor in human dendritic cells derivedfrom monocytes. Journal of cell science. 2001, vol. 114, no. pt5, p.999-1010.).

CSF-1 is also essential for proliferation and differentiation ofosteoclasts. (CECCHINI, et al. Role of CSF-1 in bone and bone marrowdevelopment. Molecular reproduction and development. 1997, vol. 46, no.1, p. 75-83.). Osteoclasts are multinucleated cells that express theCSF-1R, deriving from hematopoietic precursors that are primarilyresponsible for the degradation of mineralized bone during bonedevelopment, homeostasis and repair. In various skeletal disorders suchas osteoporosis, hypercalcemia of malignancy, rheumatoid arthritis,tumor metastases and Paget's disease, bone resorption by osteoclastsexceeds bone formation by osteoblasts leading to decreased bone mass,skeletal fragility and bone fracture. (BRUZZANITI, et al. Molecularregulation of osteoclast activity. Reviews in endocrine. 2006, vol. 7,no. 1-2, p. 123-39.). For example, patients with advanced breast cancerfrequently develop metastasis to bone. Bone metastasis results inintractable pain and a high risk of fractures due to tumor-driven boneloss (osteolysis), which is caused by increased osteoclast activity(CICEK, et al. Breast cancer bone metastasis and current smalltherapeutics. Cancer metastasis reviews. 2006, vol. 25, no. 4, p.635-44.). It has been shown that osteolysis is linked to a high level ofcirculating CSF-1 (KITAURA, et al. The journal of clinicalinvestigation. M-CSF mediates TNF-induced inflammatory osteolysis. 2005,vol. 115, no. 12, p. 3418-27.).

The CSF-1 pathway is also involved in mediating intestinal inflammationin disease such as Inflammatory bowel disease (MARSHALL, et al. Blockadeof colony stimulating factor-1 (CSF-I) leads to inhibition ofDSS-induced colitis. Inflammatory bowel diseases. 2007, vol. 13, no. 2,p. 219-24.), in mediating macrophage proliferation during acuteallograft rejection (JOSE, et al. Blockade of macrophagecolony-stimulating factor reduces macrophage proliferation andaccumulation in renal allograft rejection. American journal oftransplantation. 2003, vol. 3, no. 3, p. 294-300. and in HIV-1replication in infected macrophage (KUTZA, et al. Macrophagecolony-stimulating factor antagonists inhibit replication of HIV-1 inhuman macrophages. Journal of immunology. 2000, no. 164, p. 4955-4960.

For these reasons, the inhibition of the CSF-1 activity by variouscompounds has been proposed for the treatment of cancer and bonedegradation.

BACKGROUND ART

WO 01/30381 relates to the use of inhibitors of the CSF-1 activity inthe production of medicaments for the treatment of tumor diseases. Thetwo proposed approaches for the inhibition of the CSF-1 activity are thesuppression of the CSF-1 activity itself, and the suppression of theactivity of the CSF-1R. Neutralizing antibodies against CSF-1 or itsreceptor are preferred as the inhibitors of CSF-1 activity.

WO 03/059395 describes combination products comprising a substancecapable of inhibiting CSF-1 activity and a substance having a cytotoxicactivity for the treatment of cancer.

WO 2005/068503 discloses a method for preventing and treatingosteolysis, cancer metastasis and bone loss associated with cancermetastasis by administering an antibody against CSF-1 to a subject.

EP 1488792 A relates to the use of mono- and/or bicyclic aryl orheteroaryl quinazoline compounds which exhibit selective inhibition ofdifferentiation, proliferation or mediator release by effectivelyinhibiting CSF-1R tyrosine kinase activity. This application alsorelates to the use of such compounds for the manufacture of a medicamentfor inhibiting abnormal cell proliferation.

US 2005059113 relates to antibodies and antigen-binding portions thereofthat specifically bind to aCSF-1. The invention also relates to humananti-CSF-1 antibodies and antigen-binding portions thereof. Thisapplication invention also provides gene therapy methods using nucleicacid molecules encoding the heavy and/or light immunoglobulin moleculesthat comprise the human anti-CSF-1 antibodies.

DISCLOSURE OF INVENTION

The present invention relates to an antibody that specifically binds tothe CSF-1R.

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,unless the context clearly dictates otherwise. For example, the term “acell” includes a plurality of cells, including mixtures thereof.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange.

As used herein, the terms “comprising” and “comprise” are intended tomean that the kits of parts, products, compositions and methods includethe referenced components or steps, but not excluding others.“Consisting essentially of when used to define products, compositionsand methods, shall mean excluding other components or steps of anyessential significance. Thus, a composition consisting essentially ofthe recited components would not exclude trace contaminants andpharmaceutically acceptable carriers. “Consisting of shall meanexcluding more than trace elements of other components or steps.

As used herein, the term “specifically binds to” refers to a bindingreaction which is determinative of the presence of a target protein inthe presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated assay conditions, the antibodyaccording to the invention bind preferentially to at least part of theCSF-1R and do not bind in a significant amount to other componentspresent in a test sample. Specific binding between the antibodyaccording to the invention and the CSF-1R target means that the bindingaffinity is of at least 10³ M⁻¹, and preferably 10⁵ M⁻¹, 106 M⁻¹, 10⁷M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹ or 10¹⁹M⁻¹.

As used herein, the term “CSF-1R” refers to the human CSF1 receptor. Thehuman CSF-1 receptor has been sequenced and its amino acid sequence isdepicted in SEQ ID NO: 29.

As used herein, “antibody” or “Ab” is used in the broadest sense.Therefore, an “antibody” or “Ab” can be naturally occurring or man-madesuch as monoclonal antibodies (mAbs) produced by conventional hybridomatechnology, recombinant technology and/or a functional fragment thereof.Antibodies of the present invention are meant to include both intactimmunoglobulin molecules for example a polyclonal antibody, a monoclonalantibody (mAb), a monospecific antibody, a bispecific antibody, apolyspecific antibody, a human antibody, an animal antibody (e.g.camelid antibody), chimeric antibodies, as well as portions, fragments,regions, peptides and derivatives thereof (provided by any knowntechnique, such as, but not limited to, enzymatic cleavage, peptidesynthesis, or recombinant techniques), such as, for example,immunoglobulin devoid of light chains (see for example U.S. Pat. No.6,005,079), Fab, Fab′, F (ab′)₂, Fv, scFv, antibody fragment, diabody,Fd, CDR regions, or any portion or peptide sequence of the antibody thatis capable of binding antigen or epitope. An antibody is said to be“capable of binding” a molecule if it is capable of specificallyreacting with the molecule to thereby bind the molecule to the antibody.Antibody fragments or portions may lack the Fc fragment of intactantibody, clear more rapidly from the circulation, and may have lessnon-specific tissue binding than an intact antibody. Examples ofantibody may be produced from intact antibodies using methods well knownin the art, for example by proteolytic cleavage with enzymes such aspapain (to produce Fab fragments) or pepsin (to produce.F(ab′)₂fragments). See e.g., Wahl et al., 24 J. Nucl. Med. 316-25 (1983).Portions of antibodies may be made by any of the above methods, or maybe made by expressing a portion of the recombinant molecule. Forexample, the CDR region(s) of a recombinant antibody may be isolated andsubcloned into the appropriate expression vector.

As used herein, the term “variable region” refers to the variableregion, or domain, of the light chain (VL) or heavy chain (VH) whichcontain the determinants for binding recognition specificity. Thevariable domains are involved in antigen recognition and form theantigen binding site. As used herein, the term “framework region” refersto portions of light and heavy chain variable regions that are at least85% homologous (i.e., other than the CDR's) among different antibodiesin the same specie. As used herein, the term “homologous>> refers to acomparison of the amino acids of two polypeptides which, when aligned byusing the Smith-Waterman algorithm (SMITH, et al. Identification ofcommon molecular subsequences. Journal of Molecular Biology. 1981, no.147, p. 195-7.), have approximately the designated percentage of thesame amino acids. For example, “85% homologous” refers to a comparisonof the amino acids of two polypeptides which when optimally aligned have85% amino acid identity. The variable region of both the heavy and lightchain is divided into segments comprising four framework sub-regions(FR1, FR2, FR3, and FR4), interrupted by three stretches ofhypervariable sequences, or the complementary determining regions(CDR's), as defined in Kabat's database (Kabat et al., op. cit.), withthe CDR1 positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FR's represents two or moreof the four sub-regions constituting a framework region. The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species. The framework region of anantibody is the combined framework regions of the constituent light andheavy chains and serves to position and align the CDR's. The CDR's areprimarily responsible for forming the binding site of an antibodyconferring binding specificity and affinity to an epitope of an antigen.Within the variable regions of the H or L chains that provide for theantigen binding regions are smaller sequences dubbed “hypervariable”because of their extreme variability between antibodies of differingspecificity. Such hypervariable regions are also referred to as“complementarity determining regions” or “ ” regions. These CDR regionsaccount for the basic specificity of the antibody for a particularantigenic determinant structure. The CDRs represent non-contiguousstretches of amino acids within the variable regions but, regardless ofspecies, the positional locations of these critical amino acid sequenceswithin the variable heavy and light chain regions have been found tohave similar locations within the amino acid sequences of the variablechains. The variable heavy and light chains of all antibodies each have3 CDR regions, each non-contiguous with the others (termed L1, L2, L3,H1, H2, H3) for the respective light (L) and heavy (H) chains. Theaccepted CDR regions have been described by Kabat et al, 252 J. Biol.Chem. 6609-16 (1977), and CDR loops may be identified by applying theserules during an examination of a linear amino acid sequence. The rulesfor defining the CDR-H3 loop can vary, however (see Chapter 4, AntibodyEngineering: Methods & Protocols, (Lo, ed. Humana Press, Totowa, N.J.,2004)), and the actual boundaries of some CDR-H3 loops may not beidentified without experimental techniques such as circular dichroism,nuclear magnetic resonance, or X-ray crystallography.ln all mammalianspecies, antibody peptides contain constant (i.e., highly conserved) andvariable regions, and, within the latter, there are the CDRs and theso-called “framework regions” made up of amino acid sequences within thevariable region of the heavy or light chain but outside the CDRs. TheCDR regions can also be defined using Chothia nomenclature (CHOTHIA andLESK. Canonical structures for the hypervariable regions ofimmunoglobulins (1987) J Mol. Biol. 1987 Aug. 20; 196(4):901-17).Therefore, in certain embodiments, the CDRs are Kabat defined CDRs, andin other embodiments, the CDRs are Chothia defined CDRs. Regarding theantigenic determinate recognized by the CDR regions of the antibody,this is also referred to as the “epitope.” In other words, epitoperefers to that portion of any molecule capable of being recognized by,and bound by, an antibody (the corresponding antibody binding region maybe referred to as a paratope). In general, epitopes consist ofchemically active surface groupings of molecules, for example, aminoacids or sugar side chains, and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody that is derived from a single clone. Monoclonal antibodies canbe prepared using hybridoma techniques such as those disclosed inHARLOW. Antibodies: A Laboratory manual. 2nd edition. Cold SpringHarbor: Laboratory press, 1988. and HAMMERLING, et al. MonoclonalAntibodies and T Cell Hybridomas. New York: Elsevier, 1981. p. 563-681.

As used herein, the term “human antibody” refers to an antibody havingvariable and constant regions derived from or closely matching humangermline immunoglobulin sequences. The human antibody of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).Thus, as used herein, the term “human antibody” refers to an antibody inwhich substantially every part of the protein is substantially similarto a human germline antibody. “Substantially similar” refers to anantibody having a nucleic acid sequence which is at least 80, preferably85, more preferably 90 and even more preferably 95% homologous to thenucleic acid sequence a human germline antibody.

As used herein, the term “Fab” refers to regions of antibody moleculeswhich include the variable region of the heavy chain and light chain andwhich exhibit binding activity. “Fab” includes aggregates of one heavyand one light chain (commonly known as Fab), whether any of the aboveare covalently or non-covalently aggregated so long as the aggregationis capable of selectively reacting with a particular antigen or antigenfamily. The Fab fragment is a heterodimer comprising a VL and a secondpolypeptide comprising the VH and CH1 domains. In a preferred embodimentthe antibody is an Fab′ fragment. By Fab′ fragments differ from Fabfragments in that the Fab′ fragment contains a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody “hinge region”.

“F(ab′)₂” refers to an antibody fragment obtained by the pepsintreatment of an antibody or to the equivalent protein obtained by othertechniques such as recombinant technologies. F(ab′)₂ fragment has twoantigen-combining sites and is still capable of cross-linking anantigen.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the VH VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” or “scFv” comprise the VH and VL domains of antibody,wherein these domains are present in a single polypeptide chain.Preferably, the scFv further comprises a polypeptide linker between theVH and VL domains which enables the scFv to form the desired structurefor antigen binding (LENNARD. Standard protocols for the construction ofscFv libraries. Methods in molecular biology. 2002, no. 178, p. 59-71.).

As used herein, the term “antibody fragment” refers to one or morefragments of an antibody that retain the ability to specifically bind tothe CSF-1R.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may bind to one ore morethan one epitope. Diabodies are more fully described in POLJAK.Production and structure of diabodies. Structure. 1994, vol. 2, no. 12,p. 1121-3., HUDSON, et al. High avidity scFv multimers; diabodies andtriabodies. Journal of immunological methods. 1999, vol. 231, no. 1-2,p. 177-89. and KIPRIYANOV. Generation of bispecific and tandemdiabodies. Methods in molecular biology. 2002, no. 178, p. 317-31.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies. However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and scFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the production of large amounts of these fragments. Techniquesfor the production of antibody fragments will be apparent to the skilledin the art. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv).

As used herein “Domain Antibodies” (dAbs) consist in the smallestfunctional binding units of antibodies, corresponding to the variableregions of either the heavy (VH) or light (VL) chains of the antibodies.Domain Antibodies have a molecular weight of approximately 13 kDa, orless than one-tenth the size of a full antibody.

As used herein, “Fd” refers to an antibody fragment that consists of theVH and CH1 domains.

The term “antibody” or “Ab” also refers to other antibody fragment wellknown to the one skilled in the art, for example those described inHOLLIGER, et al. Engineered antibody fragments and the rise of singledomains. Nature biotechnology. 2005, vol. 23, no. 9, p. 1126-36. andHOOGENBOOM, et al. Natural and designer binding sites made by phagedisplay technology. Immunology today. 2000, vol. 21, no. 8, p. 371-8.

According to one embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises:

-   -   (i) at least one CDR wherein said CDR is comprising at least        five consecutive amino acids of the sequence starting in        position 45 and finishing in position 54 of SEQ ID NO:2, of the        sequence starting in position 66 and finishing in position 87 of        SEQ ID NO:2 or of the sequence starting in position 117 and        finishing in position 126 of SEQ ID NO:2;    -   or,    -   (ii) at least one CDR wherein said CDR is comprising at least        five consecutive amino acids of the sequence starting in        position 44 and finishing in position 56 of SEQ ID NO:4, of the        sequence starting in position 66 and finishing in position 76 of        SEQ ID NO:4 or of the sequence starting in position 109 and        finishing in position 117 of SEQ ID NO:4.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises at least one CDR wherein said CDRis, independently from one another, selected in the group of CDRscomprising at least five consecutive amino acids:

-   -   of the sequence starting in position 45 and finishing in        position 54 of SEQ ID NO:2,    -   of the sequence starting in position 66 and finishing in        position 87 of SEQ ID NO:2,    -   of the sequence starting in position 117 and finishing in        position 126 of SEQ ID NO:2,    -   of the sequence starting in position 44 and finishing in        position 56 of SEQ ID NO:4,    -   of the sequence starting in position 66 and finishing in        position 76 of SEQ ID NO:4    -   or of the sequence starting in position 109 and finishing in        position 117 of SEQ ID NO:4.

According to a preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises 2, 3, 4 or 5 and even morepreferably 6 CDRs wherein said CDRs are, independently from one another,selected in the group of CDRs comprising at least five consecutive aminoacids:

-   -   of the sequence starting in position 45 and finishing in        position 54 of SEQ ID NO:2,    -   of the sequence starting in position 66 and finishing in        position 87 of SEQ ID NO:2,    -   of the sequence starting in position 117 and finishing in        position 126 of SEQ ID NO:2,    -   of the sequence starting in position 44 and finishing in        position 56 of SEQ ID NO:4,    -   of the sequence starting in position 66 and finishing in        position 76 of SEQ ID NO:4    -   or of the sequence starting in position 109 and finishing in        position 117 of SEQ ID NO:4.

According to a another preferred embodiment, the antibody of theInvention binds specifically to CSF-1R and comprises:

-   -   (iii) 2 and even more preferably 3 CDRs wherein said CDRs are,        independently from one another, selected in the group of CDRs        comprising at least five consecutive amino acids:        -   of the sequence starting in position 45 and finishing in            position 54 of SEQ ID NO:2,        -   of the sequence starting in position 66 and finishing in            position 87 of SEQ ID NO:2,        -   of the sequence starting in position 117 and finishing in            position 126 of SEQ ID NO:2,    -   or    -   (iv) 2 and even more preferably 3 CDRs wherein said CDRs are,        independently from one another, selected in the group of CDRs        comprising at least five consecutive amino acids:        -   of the sequence starting in position 44 and finishing in            position 56 of SEQ ID NO:4,        -   of the sequence starting in position 66 and finishing in            position 76 of SEQ ID NO:4        -   or of the sequence starting in position 109 and finishing in            position 117 of SEQ ID NO:4.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises:

-   -   (i) at least one CDR selected, independently from one another,        in the group of the CDR as set forth in:        -   the sequence starting in position 45 and finishing in            position 54 of SEQ ID NO:2,        -   the sequence starting in position 66 and finishing in            position 87 of SEQ ID NO:2 and        -   the sequence starting in position 117 and finishing in            position 126 of SEQ ID NO:2; or    -   (ii) at least one CDR selected, independently from one another,        in the group of the CDR as set forth in:        -   the sequence starting in position 44 and finishing in            position 56 of SEQ ID NO:4,        -   the sequence starting in position 66 and finishing in            position 76 of SEQ ID NO:4 and        -   the sequence starting in position 109 and finishing in            position 117 of SEQ ID NO:4.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises at least one CDR selected,independently from one another, in the group of the CDR as set forth in:

-   -   the sequence starting in position 45 and finishing in position        54 of SEQ ID NO:2,    -   the sequence starting in position 66 and finishing in position        87 of SEQ ID NO:2,    -   the sequence starting in position 117 and finishing in position        126 of SEQ ID NO:2,    -   the sequence starting in position 44 and finishing in position        56 of SEQ ID NO:4,    -   the sequence starting in position 66 and finishing in position        76 of SEQ ID NO:4 and    -   the sequence starting in position 109 and finishing in position        117 of SEQ ID NO:4.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises 2, 3, 4 or 5 and even morepreferably 6 CDRs selected, independently from one another, in the groupof the CDR as set forth in:

-   -   the sequence starting in position 45 and finishing in position        54 of SEQ ID NO:2,    -   the sequence starting in position 66 and finishing in position        87 of SEQ ID NO:2,    -   the sequence starting in position 117 and finishing in position        126 of SEQ ID NO:2,    -   the sequence starting in position 44 and finishing in position        56 of SEQ ID NO:4,    -   the sequence starting in position 66 and finishing in position        76 of SEQ ID NO:4 and    -   the sequence starting in position 109 and finishing in position        117 of SEQ ID NO:4.

According to a another preferred embodiment, the antibody of theInvention binds specifically to CSF-1R and comprises:

-   -   (iv) 2 and even more preferably 3 CDRs wherein said CDRs are,        independently from one another, selected in the group of the        CDRs as set forth in:        -   the sequence starting in position 45 and finishing in            position 54 of SEQ ID NO:2,        -   the sequence starting in position 66 and finishing in            position 87 of SEQ ID NO:2,        -   the sequence starting in position 117 and finishing in            position 126 of SEQ ID NO:2,    -   or    -   (iv) 2 and even more preferably 3 CDRs wherein said CDRs are,        independently from one another, selected in the group of the        CDRs as set forth in:        -   the sequence starting in position 44 and finishing in            position 56 of SEQ ID NO:4,        -   the sequence starting in position 66 and finishing in            position 76 of SEQ ID NO:4,        -   the sequence starting in position 109 and finishing in            position 117 of SEQ ID NO:4.

According to one embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) at least one CDR comprising anamino acid sequence as set forth in any one of SEQ ID NOs: 11, 12, or13; or (ii) at least one CDR comprising an amino acid sequence as setforth in any one of SEQ ID NOs: 14, 15 or 16.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises at least one CDR comprising anamino acid sequence as set forth in any one of SEQ ID NOs: 11, 12, 13,14, 15 or 16.

According to a preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises 2, 3, 4, 5 and even more preferably6 CDRs comprising an amino acid sequence as set forth in any one of SEQID NOs: 11, 12, 13, 14, 15 or 16.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) at least one CDR as set forthin any one of SEQ ID NOs: 11, 12 or 13; or (ii) at least one CDR setforth in any one of SEQ ID NOs: 14, 15 or 16.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises at least one CDR as set forth inany one of SEQ ID NOs: 11, 12, 13, 14, 15 or 16.

According to one preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises 2, 3, 4, 5 and even morepreferably 6 CDRs as set forth in any one of SEQ ID NOs: 11, 12, 13, 14,or 16.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) at least one CDR comprising anamino acid sequence as set forth in any one of SEQ ID NOs: 17, 18 or 19;or (ii) at least one CDR comprising an amino acid sequence as set forthin any one of SEQ ID NOs: 20, 21 or 22.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises at least one CDR comprising anamino acid sequence as set forth in any one of SEQ ID NOs: 17, 18, 19,20, 21 or 22.

According to one preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises 2, 3, 4, 5 and even morepreferably 6 CDRs comprising an amino acid sequence as set forth in anyone of SEQ ID NOs: 17, 18, 19, 20, 21 or 22.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) at least one CDR as set forthin any one of SEQ ID NOs: 17, 18 or 19; or (ii) at least one CDR as setforth in any one of SEQ ID NOs: 20, 21 or 22.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises at least one CDR set forth in anyone of SEQ ID NOs: 17, 18, 19, 20, 21 or 22.

According to one preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises 2, 3, 4, 5 and even morepreferably 6 CDRs as set forth in any one of SEQ ID NOs: 17, 18, 19, 20,21 or 22.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) at least one CDR comprising anamino acid sequence as set forth in any one of SEQ ID NOs: 23, 24 or 25;or (ii) at least one CDR comprising an amino acid sequence as set forthin any one of SEQ ID NOs: 26, 27 or 28.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises at least one CDR comprising anamino acid sequence as set forth in any one of SEQ ID NOs: 23, 24, 25,26, 27 or 28.

According to one preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises 2, 3, 4, 5 and even morepreferably 6 CDRs comprising an amino acid sequence as set forth in anyone of SEQ ID NOs: 23, 24, 25, 26, 27 or 28.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) at least one CDR as set forthin any one of SEQ ID NOs: 23, 24 or 25; or (ii) at least one CDR as setforth in any one of SEQ ID NOs: 26, 27 or 28.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises at least one CDR as set forth inany one of SEQ ID NOs: 23, 24, 25, 26, 27 or 28.

According to one preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises 2, 3, 4, 5 and even morepreferably 6 CDRs as set forth in any one of SEQ ID NOs: 23, 24, 25, 26,27 or 28.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein saidvariable region comprises the CDRs as set forth in:

-   -   the sequence starting in position 45 and finishing in position        54 of SEQ ID NO:2,    -   the sequence starting in position 66 and finishing in position        87 of SEQ ID NO:2 and    -   the sequence starting in position 117 and finishing in position        126 of SEQ ID NO:2.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein saidvariable region comprises the CDRs as set forth in:

-   -   the sequence starting in position 44 and finishing in position        56 of SEQ ID NO:4,    -   the sequence starting in position 66 and finishing in position        76 of SEQ ID NO:4 and    -   the sequence starting in position 109 and finishing in position        117 of SEQ ID NO:4.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein saidvariable region comprises the three CDRs as set forth in SEQ ID NOs: 11,12, and 13.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein saidvariable region comprises the three CDRs as set forth in SEQ ID NOs: 14,15, and 16.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein saidvariable region comprises the three CDRs set forth in SEQ ID NOs: 17,18, and 19.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein saidvariable region comprises the three CDRs as set forth in SEQ ID NOs: 20,21, and 22.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein saidvariable region comprises the three CDRs as set forth in SEQ ID NOs: 23,24, and 25.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein saidvariable region comprises the three CDRs as set forth in SEQ ID NOs: 26,27, and 28.

In preferred embodiments, the said variable region further comprisesone, more preferably two, even more preferably three and definitelypreferably four framework region, and more preferably human FR. As usedherein, a “human FR” is a framework region that is at least 75%homologous to the framework region of a naturally occurring humanantibody.

According to one preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises a variable region, whereinthe variable region comprises an amino acid sequence as set forth in SEQID NO:6.

In a more preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein thevariable region is as set forth in SEQ ID NO:6.

In another preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises a variable region, wherein thevariable region comprises an amino acid sequence as set forth in SEQ IDNO:9.

In another more preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises a variable region, whereinthe variable region is as set forth in SEQ ID NO:9.

In another embodiment, the antibody of the Invention binds specificallyto CSF-1R and comprises two variable regions, wherein the variableregions are, independently from one another, selected in the group of

-   -   (i) variable regions comprising the CDRs as set forth in:        -   the sequence starting in position 45 and finishing in            position 54 of SEQ ID NO:2,        -   the sequence starting in position 66 and finishing in            position 87 of SEQ ID NO:2 and        -   the sequence starting in position 117 and finishing in            position 126 of SEQ ID NO:2;    -   (ii) variable regions comprising the CDR as set forth in:        -   the sequence starting in position 44 and finishing in            position 56 of SEQ ID NO:4,        -   the sequence starting in position 66 and finishing in            position 76 of SEQ ID NO:4 and        -   the sequence starting in position 109 and finishing in            position 117 of SEQ ID NO:4;    -   (ii) variable regions comprising the three CDRs set forth in SEQ        ID NOs: 11, 12, and 13;    -   (iii) variable regions comprising the three CDRs set forth in        SEQ ID NOs: 14, 15, and 16;    -   (iv) variable regions comprising the three CDRs set forth in SEQ        ID NOs: 17, 18, and 19;    -   (v) variable regions comprising the three CDRs set forth in SEQ        ID NOs: 20, 21, and 22;    -   (vi) variable regions comprising the three CDRs set forth in SEQ        ID NOs: 23, 24, and 25    -   and    -   (vii) variable regions comprising the three CDRs set forth in        SEQ ID NOs: 26, 27 and 28.

According to a preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises:

-   -   (i) a first variable region, wherein said variable region        comprises:        -   the CDR as set forth in the sequence starting in position 45            and finishing in position 54 of SEQ ID NO:2,        -   the CDR as set forth in the sequence starting in position 66            and finishing in position 87 of SEQ ID NO:2 and        -   the CDR as set forth in the sequence starting in position            117 and finishing in position 126 of SEQ ID NO:2;    -   and    -   (ii) a second variable region, wherein said variable region        comprises:        -   the CDR as set forth in the sequence starting in position 44            and finishing in position 56 of SEQ ID NO:4,        -   the CDR as set forth in the sequence starting in position 66            and finishing in position 76 of SEQ ID NO:4 and        -   the CDR as set forth in the sequence starting in position            109 and finishing in position 117 of SEQ ID NO:4.

According to a preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises:

-   -   (i) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 11, 12, and 13, and    -   (ii) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 14, 15, and 16.

According to a preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises:

-   -   (i) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 17, 18, and 19, and    -   (ii) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 20, 21, and 22.

According to a preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises:

-   -   (i) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 23, 24, and 25, and    -   (ii) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 26, 27, and 28.

According to a preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises:

-   -   (i) a variable region as set forth in SEQ ID NO:6 and    -   (ii) a variable region as set forth in SEQ ID NO:9.

According to a preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises:

-   -   (i) a heavy-chain variable region comprising:        -   the CDR as set forth in the sequence starting in position 45            and finishing in position 54 of SEQ ID NO:2,        -   the CDR as set forth in the sequence starting in position 66            and finishing in position 87 of SEQ ID NO:2 and        -   the CDR as set forth in the sequence starting in position            117 and finishing in position 126 of SEQ ID NO:2;    -   and    -   (ii) a light-chain variable region comprising:        -   the CDR as set forth in the sequence starting in position 44            and finishing in position 56 of SEQ ID NO:4,        -   the CDR as set forth in the sequence starting in position 66            and finishing in position 76 of SEQ ID NO:4 and        -   the CDR as set forth in the sequence starting in position            109 and finishing in position 117 of SEQ ID NO:4.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) a heavy-chain variable regioncomprising the three CDRs as set forth in SEQ ID NOs: 11, 12, and 13,and (ii) a light-chain variable region comprising the three CDRs as setforth in SEQ ID NOs: 14, 15, and 16.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) a heavy-chain variable regioncomprising the three CDRs as set forth in SEQ ID NOs: 17, 18, and 19,and (ii) a light-chain variable region comprising the three CDRs as setforth in SEQ ID NOs: 20, 21, and 22.

According to another embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) a heavy-chain variable regioncomprising the three CDRs as set forth in SEQ ID NOs: 23, 24, and 25,and (ii) a light-chain variable region comprising the three CDRs as setforth in SEQ ID NOs: 26, 27, and 28.

According to a preferred embodiment, the antibody of the Invention bindsspecifically to CSF-1R and comprises (i) a heavy-chain variable regionas set forth in SEQ ID NO:6 and (ii) a light-chain variable region asset forth in SEQ ID NO:9.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises:

-   -   (i) a variable regions comprising:        -   the CDR as set forth in the sequence starting in position 45            and finishing in position 54 of SEQ ID NO:2,        -   the CDR set forth in the sequence starting in position 66            and finishing in position 87 of SEQ ID NO:2 and        -   the CDR set forth in the sequence starting in position 117            and finishing in position 126 of SEQ ID NO:2;    -   and    -   (ii) a variable region comprising:        -   the CDR set forth in the sequence starting in position 44            and finishing in position 56 of SEQ ID NO:4,        -   the CDR set forth in the sequence starting in position 66            and finishing in position 76 of SEQ ID NO:4 and        -   the CDR set forth in the sequence starting in position 109            and finishing in position 117 of SEQ ID NO:4.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises:

-   -   (i) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 11, 12, and 13, and    -   (ii) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 14, 15, and 16.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises:

-   -   (i) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 17, 18, and 19, and    -   (ii) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 20, 21, and 22.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises

-   -   (i) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 23, 24, and 25, and    -   (ii) a variable region comprising the three CDRs as set forth in        SEQ ID NOs: 26, 27, and 28.

According to a more preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises:

-   -   (i) a variable region as set forth in SEQ ID NO:6 and    -   (ii) a variable region as set forth in SEQ ID NO:9.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises:

-   -   (i) a heavy-chain variable region comprising:        -   the CDR set forth in the sequence starting in position 45            and finishing in position 54 of SEQ ID NO:2,        -   the CDR set forth in the sequence starting in position 66            and finishing in position 87 of SEQ ID NO:2 and        -   the CDR set forth in the sequence starting in position 117            and finishing in position 126 of SEQ ID NO:2    -   and    -   (ii) a light-chain variable region comprising:        -   the CDR set forth in the sequence starting in position 44            and finishing in position 56 of SEQ ID NO:4,        -   the CDR set forth in the sequence starting in position 66            and finishing in position 76 of SEQ ID NO:4 and        -   the CDR set forth in the sequence starting in position 109            and finishing in position 117 of SEQ ID NO:4.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises:

-   -   (i) a heavy-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 11, 12, and 13, and    -   (ii) a light-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 14, 15, and 16.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises:

-   -   (i) a heavy-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 17, 18, and 19, and    -   (ii) a light-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 20, 21, and 22.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises:

-   -   (i) a heavy-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 23, 24, and 25, and    -   (ii) a light-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 26, 27, and 28.

According to a more preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and is a scFv, wherein said scFv comprises:

-   -   (i) the heavy-chain variable region as set forth in SEQ ID NO:6        and    -   (ii) the light-chain variable region as set forth in SEQ ID        NO:9.

In an even more preferred embodiment, the scFv is provided wherein atleast one amino acid is substituted (according to Table 1 and Table 2)within the amino acid sequence as set forth in SEQ ID NO 6 and 9. In adefinitely preferred embodiment the human antibody is provided whereinall the amino acid depicted in Table 1 and Table 2 are substituted(according to Table 1 and Table 2) within the amino acid sequence as setforth in SEQ ID NO 6 and 9.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises the heavy chain as set forthin SEQ ID NO:2.

According to another preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises the light chain as set forthin SEQ ID NO:4.

According to a more preferred embodiment, the antibody of the Inventionbinds specifically to CSF-1R and comprises the heavy chain as set forthin SEQ ID NO:2 and the light chain as set forth in SEQ ID NO:4.

According to an even more preferred embodiment, the antibody of theInvention binds specifically to CSF-1R and comprises two heavy chains asset forth in SEQ ID NO:2 and two light chain as set forth in SEQ IDNO:4. This particular antibody will be named CXIIG6 throughout thepresent application.

According to another preferred embodiment, the present invention relatesto a human antibody, that specifically binds to CSF-1R, comprising:

-   -   (i) a heavy-chain variable region comprising:        -   the CDR as set forth in the sequence starting in position 45            and finishing in position 54 of SEQ ID NO:2,        -   the CDR as set forth in the sequence starting in position 66            and finishing in position 87 of SEQ ID NO:2 and        -   the CDR as set forth in the sequence starting in position            117 and finishing in position 126 of SEQ ID NO:2;    -   and    -   (ii) a light-chain variable region comprising:        -   the CDR as set forth in the sequence starting in position 44            and finishing in position 56 of SEQ ID NO:4,        -   the CDR as set forth in the sequence starting in position 66            and finishing in position 76 of SEQ ID NO:4 and        -   the CDR as set forth in the sequence starting in position            109 and finishing in position 117 of SEQ ID NO:4.

According to another embodiment, the present invention relates to anhuman antibody, that specifically binds to CSF-1R, comprising:

-   -   (i) a heavy-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 11, 12, and 13, and    -   (ii) a light-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 14, 15, and 16.

According to another embodiment, the present invention relates to anhuman antibody, that specifically binds to CSF-1R, comprising:

-   -   (i) a heavy-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 17, 18, and 19, and    -   (ii) a light-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 20, 21, and 22.

According to another embodiment, the present invention relates to anhuman antibody, that specifically binds to CSF-1R, comprising:

-   -   (i) a heavy-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 23, 24, and 25, and    -   (ii) a light-chain variable region comprising the three CDRs as        set forth in SEQ ID NOs: 26, 27, and 28.

According to a preferred embodiment, the present invention relates to anhuman antibody, that specifically binds to CSF-1R, comprising:

-   -   (i) the heavy-chain variable region set forth in SEQ ID NO:6 and    -   (ii) the light-chain variable region set forth in SEQ ID NO:9.

In a more preferred embodiment, the human antibody is provided whereinat least one amino acid is substituted (according to Table 1 and Table2) within the amino acid sequence as set forth in SEQ ID NO: 6 and 9. Ina even more preferred embodiment the human antibody is provided whereinall the amino acid depicted in Table 1 and Table 2 are substituted(according to Table 1 and Table 2) within the amino acid sequence as setforth in SEQ ID NO: 6 and 9.

TABLE 1 SEQ ID NO: 6 position Preferred substitution K 3 Q E 5 V M 18 LK 19 R W 33 Y S 40 A E 42 G M 43 K A 49 G E 50 R I 51 T A 59 T F 61 E E64 A S 79 N V 81 L R 89 K P 90 T, A G 94 A I 95 V T 99 A N 107 Y, V T113 L L 114 V

TABLE 2 SEQ ID NO: 9 Position Preferred substitution A 9 S V 13 A E 17 DT 18 R E 27 Q N 28 G Q 40 P, D S 43 A Q 45 K V 48 L H 49 Y N 53 R A 55 ED 56 S Q 70 D Y 71 F S 72 T K 74 T N 76 S S 80 P G 84 A S 85 T H 90 Q G100 Q

The antibody, more specifically the human antibody, according to theinvention may be of different isotypes, such as IgG, IgA, IgM or IgE. Ina preferred embodiment the antibody, more specifically the humanantibody, according to the invention is an IgG.

In a related embodiment, the human antibody comprises a modified orunmodified constant region of a human IgGI, IgG2, IgG3 or IgG4. In apreferred embodiment, the constant region is human IgG1 or IgG4, whichmay optionally be modified to enhance or decrease certain properties.

In the case of IgG1, modifications to the constant region, particularlythe hinge or CH2 region, may increase or decrease effector function,including ADCC and/or CDC activity. In other embodiments, an IgG2constant region is modified to decrease antibody-antigen aggregateformation. In the case of IgG4, modifications to the constant region,particularly the hinge region, may reduce the formation ofhalf-antibodies.

The desired binding affinity may be retained even though one or more ofthe amino acids in the antibody are mutated. These variants have atleast one amino acid in the antibody replaced by a different residue.According to another embodiment, the present invention provides anantibody that specifically binds to CSF1 as above described in which atleast one of the amino acid comprised in the CDR(s) is conservativelysubstituted. Conservative substitutions are shown in Table 3.

TABLE 3 Original Amino Preferred conservative More preferredconservative Acid substitution substitution A V, L, I V R K, Q, N K N Q,H, D, K, R Q D E, N E C S, A S Q N, E N E D, Q D G A A H N, Q, K, R R IL, V, M, A, F L L I, V, M, A, F I K R, Q, N R M L, F, I L F W, L, V, I,A, Y Y P A A S T T T V, S S W Y, F Y Y W, F, T, S F V L, M, F, A L

The present invention also relates to a process of modifying theantibody of the invention by affinity maturation.

As used herein, “affinity maturation” refers to the substitution of oneor more amino acid comprised in one or more CDRs, said substitutionresulting in an improvement in the affinity of the antibody to CSF-1R,compared to a parent antibody which does not possess thosesubstitution(s). Affinity maturation processes are known in the art. Seefor example methods disclosed in MARKS, et al. By-passing immunization:building high affinity human antibodies by chain shuffling.Biotechnology. 1992, vol. 10, no. 7, p. 779-83.; BARBAS, et al. In vitroevolution of a neutralizing human antibody to human immunodeficiencyvirus type 1 to enhance affinity and broaden strain cross-reactivity.Proceedings of the National Academy of Sciences of the United States ofAmerica. 1994, vol. 91, no. 9, p. 3809-13.; SCHIER. Identification offunctional and structural amino-acid residues by parsimoniousmutagenesis. Gene. 1996, vol. 169, no. 2, p. 147-55.; YELTON. Affinitymaturation of the BR96 anti-carcinoma antibody by codon-basedmutagenesis. J. immunol. 1995, vol. 155, no. 4, p. 1994-2004.; JACKSON,et al. In vitro antibody maturation. Improvement of a high affinity,neutralizing antibody against IL-1 beta. J. immunol. 1995, vol. 154, no.7, p. 3310-9. and HAWKINS, et al. Selection of phage antibodies bybinding affinity. Mimicking affinity maturation. Journal of molecularbiology. 1992, vol. 226, no. 3, p. 889-96.

The present invention also relates to an antibody, that specificallybinds to CSF-1R, obtained by affinity maturation as previouslydescribed.

In another embodiment, the present invention provides variants of theantibody previously described, having an amino acid sequence which is atleast 80%, preferably at least 85%, more preferably at least 90%, andeven more preferably at least 98% homologous to the amino acid sequenceof the previously described antibody.

In another embodiment, the antibody according to the inventionspecifically binds to more than one epitope. For example, the antibodyaccording to the invention may bind to two different epitopes of CSF-1R.Alternatively, the antibody according to the invention can be able tobind to CSF-1R and to another molecule. As used herein, antibodies thatspecifically bind to more than one epitope can be cross-linkedantibodies. For example, an antibody can be coupled to avidin, the otherto biotin. Cross-linked antibodies may be made using any convenientcross-linking methods well known in the art. Techniques for generatingbispecific antibodies from antibody fragments have also been described,see for example BRENNAN, et al. Preparation of bispecific antibodies bychemical recombination of monoclonal immunoglobulin G1 fragments.Science. 1985, vol. 229, no. 4708, p. 81-3. and SHALABY, et al.Development of humanized bispecific antibodies reactive with cytotoxiclymphocytes and tumor cells overexpressing the HER2 protooncogene. TheJournal of experimental medicine. 1992, vol. 175, no. 1, p. 217-25.KOSTELNY, et al. Formation of a bispecific antibody by the use ofleucine zippers. J. immunol. 1992, vol. 148, no. 5, p. 1547-33.

According to a preferred embodiment, the antibody that specificallybinds to more than one epitope according to the invention is a diabody.

According to another preferred embodiment, the antibody thatspecifically binds to more than one epitope according to the inventionis a linear antibody as described in ZAPATA, et al. Engineering linearF(ab′)2 fragments for efficient production in Escherichia coli andenhanced antiproliferative activity. Protein engineering. 1995, vol. 8,no. 10, p. 1057-62.

The antibody according to the invention may be glycosylated ornon-glycosylated.

As used herein, the term “glycosylation” refers to the presence ofcarbohydrate units that are covalently attached to the antibody.

In another embodiment, the antibody according to the invention isconjugated to a radiosensitizer agent, a receptor and/or a cytotoxicagent

As used herein, the term “radiosensitizer” refers to a molecule thatmakes cells more sensitive to radiation therapy. Radiosensitizerincludes, but are not limited to, metronidazole, misonidazole,desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycinC, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine(BUdR), iododeoxyuridine(IUdR), bromodeoxycytidine, fluorodeoxyuridine(FUdR), hydroxyurea and cisplatin.

As used herein, the term “receptor” refers to a compound able tospecifically bind to a ligand. According to a preferred embodiment ofthe invention, the receptor is biotin.

As used herein, the term cytoxic agent refers to a compound that isdirectly toxic to cells, preventing their reproduction or growth.According to a preferred embodiment, the cytotoxic agent used in thecontext of the present invention is chosen from the group comprisingcancer therapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope.

In another embodiment, the antibody according to the invention isconjugated to a labelling agent

As used herein, “a labelling agent” refers to a detectable compound. Thelabelling agent may be detectable by itself (e.g., radioisotope labelsor fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical modification of a substrate compound which isdetectable.

As used herein, the term “conjugated” means that the antibody accordingto the invention and the labelling agent are covalently ornon-covalently linked.

“Covalent link” refers to coupling through reactive functional groups,optionally with the intermediary use of a cross linker or otheractivating agent (see for example HERMANSON. Bioconjugate techniques.Academic press, 1996.). The antibody according to the invention and/orthe conjugated agent may be modified in order to allow their couplingvia, for example, substitution on an activated carbonyl group (includingthose activated in situ) or on an imidoester, via addition on anunsaturated carbonyl group, by reductive amination, nucleophilicsubstitution on a saturated carbon atom or on a heteroatom, by reactionon aromatic cycles, In particular, coupling may be done usinghomobifunctional or heterobifunctional cross-linking reagents.Homobifunctional cross linkers including glutaraldehyde, succinic acidand bis-imidoester like DMS (dimethyl suberimidate) can be used tocouple amine groups which may be present on the various moieties.Numerous examples are given in HERMANSON. Bioconjugate techniques.Academic press, 1996. p. 118-228. which are well known by those of theart. Heterobifunctional cross linkers include those having both aminereactive and sulfhydryl-reactive groups, carbonyl-reactive andsulfhydryl-reactive groups and sulfhydryl-reactive groups andphotoreactive linkers. Suitable heterobifunctional cross-linkers are,for example, described in HERMANSON. Bioconjugate techniques. Academicpress, 1996. p. 229-285. Examples are, for example, SPDP(N-succinimidyl3-(2-pyridyldithio) propionate), SMBP(succinimidyl-4-(p-maleimidophenyl) butyrate), SMPT(succinimidyloxycarbonyl-methyl-(-2-pyridyldithio) toluene), MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester), STAB (N-succinimidyl (4iodoacetyl)aminobenzoate), GMBS (γ-maleimidobutyryloxy) succinimideester), SIAX (succinimidyl-6-iodoacetyl amino hexonate, SIAC(succinimidyl-4-iodoacetyl amino methyl), NPIA (p-nitrophenyliodoacetate). Other examples are useful to couplecarbohydrate-containing molecules (e.g. env glycoproteins, antibodies)to sulfydryl-reactive groups. Examples include MPBH (4-(4-Nmaleimidophenyl) butyric acid hydrazide) and PDPH(4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (M2C2H and3-2(2-pyridyldithio) proprionyl hydrazide).

According to another embodiment, the present invention relates to anucleic acid sequence coding the antibody of the invention.

The term “nucleic acid sequence” refers to a linear sequence ofnucleotides. The nucleotides are either a linear sequence ofpolyribonucleotides or polydeoxyribonucleotides, or a mixture of both.Examples of polynucleotides in the context of the present inventioninclude single and double stranded DNA, single and double stranded RNA,and hybrid molecules that have both mixtures of single and doublestranded DNA and RNA. Further, the polynucleotides of the presentinvention may have one or more modified nucleotides.

According to a preferred embodiment of the invention, the nucleic acidsequence according to the invention is comprised in a vector.

The vector can be of plasmid or viral origin and can, where appropriate,be combined with one or more substances which improve the transfectionalefficiency and/or stability of the vector. These substances are widelydocumented in the literature which is available to the skilled person(see, for example, FELGNER, et al. Cationic liposome mediatedtransfection. Proceedings of the Western Pharmacology Society. 1989,vol. 32, p. 115-21.; HODGSON, et al. Virosomes: cationic liposomesenhance retroviral transduction. Nature biotechnology. 1996, vol. 14,no. 3, p. 339-42.; REMY, et al. Gene transfer with a series oflipophilic DNA-binding molecules. Bioconjugate chemistry. 1994, vol. 5,no. 6, p. 647-54.). By way of non-limiting illustration, the substancescan be polymers, lipids, in particular cationic lipids, liposomes,nuclear proteins or neutral lipids. These substances can be used aloneor in combination. A combination which can be envisaged is that of arecombinant plasmid vector which is combined with cationic lipids (DOGS,DC-CHOL, spermine-chol, spermidine-chol, etc.), lysophospholipides (forexample Hexadecylphosphocholine) and neutral lipids (DOPE).

According to a preferred embodiment, the cationic lipids which can beused in the present invention are the cationic lipids describes in EP901463 B and more preferably pcTG90.

The choice of the plasmids which can be used within the context of thepresent invention is huge. They can be cloning vectors and/or expressionvectors. In a general manner, they are known to the skilled person and,while a number of them are available commercially, it is also possibleto construct them or to modify them using the techniques of geneticmanipulation. Examples which may be mentioned are the plasmids which arederived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript(Stratagene), pREP4, pCEP4 (Invitrogene) or p Poly (LATHE, et al.Plasmid and bacteriophage vectors for excision of intact inserts. Gene.1987, vol. 57, no. 2-3, p. 193-201.). Preferably, a plasmid which isused in the context of the present invention contains an origin ofreplication which ensures that replication is initiated in a producercell and/or a host cell (for example, the ColE1 origin will be chosenfor a plasmid which is intended to be produced in E. coli and theoriP/EBNA1 system will be chosen if it desired that the plasmid shouldbe self-replicating in a mammalian host cell, LUPTON, et al. Mappinggenetic elements of Epstein-Barr virus that facilitate extrachromosomalpersistence of Epstein-Barr virus-derived plasmids in human cells.Molecular and cellular biology. 1985, vol. 5, no. 10, p. 2533-42.;YATES, et al. Stable replication of plasmids derived from Epstein-Barrvirus in various mammalian cells. Nature. 1985, vol. 313, no. 6005, p.812-5.). The plasmid can additionally comprise a selection gene whichenables the transfected cells to be selected or identified(complementation of an auxotrophic mutation, gene encoding resistance toan antibiotic, etc.). Naturally, the plasmid can contain additionalelements which improve its maintenance and/or its stability in a givencell (cer sequence, which promotes maintenance of a plasmid in monomericform (SUMMERS, et al. Multimerization of high copy number plasmidscauses instability: ColE1 encodes a determinant essential for plasmidmonomerization and stability. Cell. 1984, vol. 36, no. 4, p. 1097-103.,sequences for integration into the cell genome).

With regard to a viral vector, it is possible to envisage a vector whichis derived from a poxvirus (vaccinia virus, in particular MVA,canarypoxvirus, etc.), from an adenovirus, from a retrovirus, from aherpesvirus, from an alphavirus, from a foamy virus or from anadenovirus-associated virus. It is possible to use replication competentor replication deficient viral vectors. Preference will be given tousing a vector which does not integrate. In this respect, adenoviralvectors and vectors deriving from poxvirus and more preferably vacciniavirus and MVA are very particularly suitable for implementing thepresent invention.

According to a preferred embodiment, the viral vector according to theinvention derives from a Modified Vaccinia Virus Ankara (MVA). MVAvectors and methods to produce such vectors are fully described inEuropean patents EP 83286 A and EP 206920 A, as well as in SUTTER, etal. Nonreplicating vaccinia vector efficiently expresses recombinantgenes. Proc. Natl. Acad. Sci. U.S.A. 1992, vol. 89, no. 22, p. 10847-51.According to a more preferred embodiment, the nucleic acid sequenceaccording to the invention may be inserted in deletion I, II, III, IV, Vand VI of the MVA vector and even more preferably in deletion III(MEYER, et al. Mapping of deletions in the genome of the highlyattenuated vaccinia virus MVA and their influence on virulence. TheJournal of general virology. 1991, vol. 72, no. Pt5, p. 1031-8.; SUTTER,et al. A recombinant vector derived from the host range-restricted andhighly attenuated MVA strain of vaccinia virus stimulates protectiveimmunity in mice to influenza virus. Vaccine. 1994, vol. 12, no. 11, p.1032-40.).

Retroviruses have the property of infecting, and in most casesintegrating into, dividing cells and in this regard are particularlyappropriate for use in relation to cancer. A recombinant retrovirusaccording to the invention generally contains the LTR sequences, anencapsidation region and the nucleotide sequence according to theinvention, which is placed under the control of the retroviral LTR or ofan internal promoter such as those described below. The recombinantretrovirus can be derived from a retrovirus of any origin (murine,primate, feline, human, etc.) and in particular from the MoMuLV (Moloneymurine leukemia virus), MVS (Murine sarcoma virus) or Friend murineretrovirus (Fb29). It is propagated in an encapsidation cell line whichis able to supply in trans the viral polypeptides gag, pol and/or envwhich are required for constituting a viral particle. Such cell linesare described in the literature (PA317, Psi CRIP GP+Am-12 etc.). Theretroviral vector according to the invention can contain modifications,in particular in the LTRs (replacement of the promoter region with aeukaryotic promoter) or the encapsidation region (replacement with aheterologous encapsidation region, for example the VL3O type) (see U.S.Pat. No. 5,747,323)

Preference will be also given to using an adenoviral vector which lacksall or part of at least one region which is essential for replicationand which is selected from the E1, E2, E4 and L1-L5 regions in order toavoid the vector being propagated within the host organism or theenvironment. A deletion of the E1 region is preferred. However, it canbe combined with (an)other modification(s)-/deletion(s) affecting, inparticular, all or part of the E2, E4 and/or L1-L5 regions, to theextent that the defective essential functions are complemented in transby means of a complementing cell line and/or a helper virus. In thisrespect, it is possible to use second-generation vectors of the state ofthe art (see, for example, international applications WO 94/28152 and WO97/04119). By way of illustration, deletion of the major part of the E1region and of the E4 transcription unit is very particularlyadvantageous. For the purpose of increasing the cloning capacities, theadenoviral vector can additionally lack all or part of the non-essentialE3 region. According to another alternative, it is possible to make useof a minimal adenoviral vector which retains the sequences which areessential for encapsidation, namely the 5′ and 3′ ITRs (InvertedTerminal Repeat), and the encapsidation region. The various adenoviralvectors, and the techniques for preparing them, are known (see, forexample, GRAHAM, et al. Methods in molecular biology. Edited by MURREY.The human press inc, 1991. p. 109-128.).

Furthermore, the origin of the adenoviral vector according to theinvention can vary both from the point of view of the species and fromthe point of view of the serotype. The vector can be derived from thegenome of an adenovirus of human or animal (canine, avian, bovine,murine, ovine, porcine, simian, etc.) origin or from a hybrid whichcomprises adenoviral genome fragments of at least two different origins.More particular mention may be made of the CAV-I or CAV-2 adenovirusesof canine origin, of the DAV adenovirus of avian origin or of the Badtype 3 adenovirus of bovine origin (ZAKHARCHUK, et al. Physical mappingand homology studies of egg drop syndrome (EDS-76) adenovirus DNA.Archives of virology. 1993, vol. 128, no. 1-2, p. 171-6.; SPIBEY, et al.Molecular cloning and restriction endonuclease mapping of two strains ofcanine adenovirus type 2. The Journal of general virology. 1989, vol.70, no. Pt 1, p. 165-72.; JOUVENNE, et al. Cloning, physical mapping andcross-hybridization of the canine adenovirus types 1 and 2 genomes.Gene. 1987, vol. 60, no. 1, p. 21-8.; MITTAL, et al. Development of abovine adenovirus type 3-based expression vector. The Journal of generalvirology. 1995, vol. 76, no. Pt 1, p. 93-102.). However, preference willbe given to an adenoviral vector of human origin which is preferablyderived from a serotype C— adenovirus, in particular a type 2 or 5serotype C adenovirus.

The term “replication-competent” as used herein refers to a viral vectorcapable of replicating in a host cell in the absence of anytrans-complementation.

According to a preferred embodiment of the invention, the replicationcompetent vector is a replication competent adenoviral vector. Thesereplication competent adenoviral vectors are well known by the oneskilled in the art. Among these, adenoviral vectors deleted in the E1bregion coding the 55 kD P53 inhibitor, as in the ONYX-015 virus(BISCHOFF, et al. An adenovirus mutant that replicates selectively inp53-deficient human tumor cells. Science. 1996, vol. 274, no. 5286, p.373-6.; He HEISE, et al. An adenovirus EIA mutant that demonstratespotent and selective systemic anti-tumoral efficacy. Nature Medicine.2000, vol. 6, no. 10, p. 1134-9.; WO 94/18992), are particularlypreferred. Accordingly, this virus can be used to selectively infect andkill p53-deficient neoplastic cells. A person of ordinary skill in theart can also mutate and disrupt the p53 inhibitor gene in adenovirus 5or other viruses according to established techniques. Adenoviral vectorsdeleted in the EIA Rb binding region can also be used in the presentinvention. For example, Delta24 virus which is a mutant adenoviruscarrying a 24 base pair deletion in the EIA region (FUEYO, et al. Amutant oncolytic adenovirus targeting the Rb pathway producesanti-glioma effect in vivo. Oncogene. 2000, vol. 19, no. 1, p. 2-12.).Delta24 has a deletion in the Rb binding region and does not bind to Rb.Therefore, replication of the mutant virus is inhibited by Rb in anormal cell. However, if Rb is inactivated and the cell becomesneoplastic, Delta24 is no longer inhibited. Instead, the mutant virusreplicates efficiently and lyses the Rb-deficient cell.

An adenoviral vector according to the present invention can be generatedin vitro in Escherichia coli (E. coli) by ligation or homologousrecombination (see, for example, international application WO 96/17070)or else by recombination in a complementing cell line.

According to a preferred embodiment of the invention, the vector furthercomprises the elements necessary for the expression of the antibodyaccording to the invention.

The elements necessary for the expression consist of all the elementswhich enable the nucleic acid sequence to be transcribed into RNA andthe mRNA to be translated into polypeptide. These elements comprise, inparticular, a promoter which may be regulable or constitutive.Naturally, the promoter is suited to the chosen vector and the hostcell. Examples which may be mentioned are the eukaryotic promoters ofthe PGK (phosphoglycerate kinase), MT (metallothionein; MCIVOR. Humanpurine nucleoside phosphorylase and adenosine deaminase: gene transferinto cultured cells and murine hematopoietic stem cells by usingrecombinant amphotropic retroviruses. Molecular and cellular biology.1987, vol. 7, no. 2, p. 838-46.), α-1 antitrypsin, CFTR, surfactant,immunoglobulin, actin (TABIN, et al. Adaptation of a retrovirus as aeucaryotic vector transmitting the herpes simplex virus thymidine kinasegene. Molecular and cellular biology. 1982, vol. 2, no. 4, p. 426-36.)and SRa (TAKEBE, et al. SR alpha promoter: an efficient and versatilemammalian cDNA expression system composed of the simian virus 40 earlypromoter and the R-U5 segment of human T-cell leukemia virus type 1 longterminal repeat. Molecular and cellular biology. 1988, vol. 8, no. 1, p.466-72.) genes, the early promoter of the SV40 virus (Simian virus), theLTR of RSV (Rous sarcoma virus), the HSV-I TK promoter, the earlypromoter of the CMV virus (Cytomegalovirus), the p7.5K pH5R, pK1L, p28and p11 promoters of the vaccinia virus, chimeric promoters such asp11K7.5 and the E1A and MLP adenoviral promoters. The promoter can alsobe a promoter which stimulates expression in a tumor or cancer cell.Particular mention may be made of the promoters of the MUC-I gene, whichis overexpressed in breast and prostate cancers (CHEN, et al. Breastcancer selective gene expression and therapy mediated by recombinantadenoviruses containing the DF3/MUC1 promoter. The Journal of clinicalinvestigation. 1995, vol. 96, no. 6, p. 2775-82.), of the CEA (standingfor carcinoma embryonic antigen) gene, which is overexpressed in coloncancers (SCHREWE, et al. Cloning of the complete gene forcarcinoembryonic antigen: analysis of its promoter indicates a regionconveying cell type-specific expression. Molecular and cellular biology.1990, vol. 10, no. 6, p. 2738-48.) of the tyrosinase gene, which isoverexpressed in melanomas (VILE, et al. Use of tissue-specificexpression of the herpes simplex virus thymidine kinase gene to inhibitgrowth of established murine melanomas following direct intratumoralinjection of DNA. Cancer res. 1993, vol. 53, no. 17, p. 3860-4.), of theERBB-2 gene, which is overexpressed in breast and pancreatic cancers(HARRIS, et al. Gene therapy for cancer using tumour-specific prodrugactivation. Gene therapy. 1994, vol. 1, no. 3, p. 170-5.) and of theα-fetoprotein gene, which is overexpressed in liver cancers (KANAI, etal. In vivo gene therapy for alpha-fetoprotein-producing hepatocellularcarcinoma by adenovirus-mediated transfer of cytosine deaminase gene.Cancer res. 1997, vol. 57, no. 3, p. 461-5.). The cytomegalovirus (CMV)early promoter is very particularly preferred.

However, when a vector deriving from a Vaccinia Virus (as for example anMVA vector) is used, the promoter of the thymidine kinase 7.5K gene andthe pH5R promoter are particularly preferred.

The necessary elements can furthermore include additional elements whichimprove the expression of the nucleic acid sequence according to theinvention or its maintenance in the host cell. Intron sequences,secretion signal sequences, nuclear localization sequences, internalsites for the reinitiation of translation of IRES type, transcriptiontermination poly

A sequences, tripartite leaders and origins of replication may inparticular be mentioned. These elements are known to the skilled person.Among secretion signal sequence, sequences encoding the polypeptides asset forth in SEQ ID NO 5 and/or 8 are particularly preferred.

The recombinant vector according to the invention can also comprise oneor more additional genes of interest, with it being possible for thesegenes to be placed under the control of the same regulatory elements(polycistronic cassette) or of independent elements. Genes which may inparticular be mentioned are the genes encoding interleukins IL-2, IL-4,IL-7, IL-10, IL-12, IL-15, IL-18, chemokines as CCL19, CCL20, CCL21,CXCL-14, interferons, tumor necrosis factor (TNF), and factors acting oninnate immunity and angiogenesis (for example PAI-1, standing forplasminogen activator inhibitor). In one particular embodiment, therecombinant vector according to the invention comprises the gene ofinterest encoding IL-2.

The present invention also relates to a cell comprising the nucleic acidsequence according to the invention. In a preferred embodiment, he cellaccording to the invention is eukaryotic cell and more preferably amammalian cell. Mammalian cells available as hosts for expression arewell known in the art and include many immortalized cell lines, such asbut not limited to, Chinese Hamster Ovary (CHO) cells, Baby HamsterKidney (BHK) cells and many others. Suitable additional eukaryotic cellsinclude yeast and other fungi.

The present invention also relates to a process for producing anantibody according to the invention comprising culturing the cellaccording to the invention under conditions permitting expression of theantibody and purifying the antibody from the cell or medium surroundingthe cell.

In another embodiment, the present invention relates to a pharmaceuticalcomposition comprising any one of the antibody, the nucleic acidsequence or the vector according to the invention and a pharmaceuticallyacceptable carrier. In a preferred embodiment, the pharmaceuticalcomposition further comprises a compound of interest.

The pharmaceutically acceptable carrier is preferably isotonic,hypotonic or weakly hypertonic and has a relatively low ionic strength,such as for example a sucrose solution. Moreover, such a carrier maycontain any solvent, or aqueous or partially aqueous liquid such asnonpyrogenic sterile water. The pH of the pharmaceutical composition is,in addition, adjusted and buffered so as to meet the requirements of usein vivo. The pharmaceutical composition may also include apharmaceutically acceptable diluent, adjuvant or excipient, as well assolubilizing, stabilizing and preserving agents. For injectableadministration, a formulation in aqueous, nonaqueous or isotonicsolution is preferred. It may be provided in a single dose or in amultidose in liquid or dry (powder, lyophilisate and the like) formwhich can be reconstituted at the time of use with an appropriatediluent.

The present invention also relates, to a kit of part comprising (i) apharmaceutical composition, an antibody, a nucleic acid sequence or avector according to the invention and, (ii) a compound of interest.

As used herein the term, “compound of interest” relates to a therapeuticcompound and preferably to a cancer therapeutic agent or a compounduseful in the treatment of bone mass decrease.

According to a preferred embodiment, the cancer therapeutic agent ischosen from the group comprising Abraxane (Paclitaxel Albumin-stabilizedNanoparticle Formulation), Adriamycin (Doxorubicin Hydrochloride),Adrucil (Fluorouracil), Aldara (Imiquimod), Alemtuzumab, Alimta(Pemetrexed Disodium), Aminolevulinic Acid, Anastrozole, Aprepitant,Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine),Arsenic Trioxide, Avastin (Bevacizumab), Azacitidine, Bevacizumab,Bexarotene, Bortezomib, Campath (Alemtuzumab), Camptosar (IrinotecanHydrochloride), Capecitabine, Carboplatin, Cetuximab, Cisplatin, Clafen(Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar(Clofarabine), Cyclophosphamide, Cytarabine, Cytosar-U (Cytarabine),Cytoxan (Cyclophosphamide), Dacogen (Decitabine), Dasatinib, Decitabine,DepoCyt (Liposomal Cytarabine), DepoFoam (Liposomal Cytarabine),Dexrazoxane Hydrochloride, Docetaxel, Doxil (Doxorubicin HydrochlorideLiposome), Doxorubicin Hydrochloride, Doxorubicin HydrochlorideLiposome, Dox-SL (Doxorubicin Hydrochloride Liposome), Efudex(Fluorouracil), Ellence (Epirubicin Hydrochloride), Eloxatin(Oxaliplatin), Emend (Aprepitant), Epirubicin Hydrochloride, Erbitux(Cetuximab), Erlotinib Hydrochloride, Evacet (Doxorubicin HydrochlorideLiposome), Evista (Raloxifene Hydrochloride), Exemestane, Faslodex(Fulvestrant), Femara (Letrozole), Fluoroplex (Fluorouracil),Fluorouracil, Fulvestrant, Gefitinib, Gemcitabine Hydrochloride,Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gleevec(Imatinib Mesylate), Herceptin (Trastuzumab), Hycamtin (TopotecanHydrochloride), Imatinib Mesylate, Imiquimod, Iressa (Gefitinib),Irinotecan Hydrochloride, Ixabepilone, Ixempra (Ixabepilone), Keoxifene(Raloxifene Hydrochloride), Kepivance (Palifermin), LapatinibDitosylate, Lenalidomide, Letrozole, Levulan (Aminolevulinic Acid),LipoDox (Doxorubicin Hydrochloride Liposome), Liposomal Cytarabine,Methazolastone (Temozolomide), Methotrexate, Mylosar (Azacitidine),Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (PaclitaxelAlbumin-stabilized Nanoparticle Formulation), Nelarabine, Neosar(Cyclophosphamide), Nexavar (Sorafenib Tosylate), Nilotinib, Nolvadex(Tamoxifen Citrate), Oncaspar (Pegaspargase), Oxaliplatin, Paclitaxel,Paclitaxel Albumin-stabilized Nanoparticle Formulation, Palifermin,Panitumumab, Paraplat (Carboplatin), Paraplatin (Carboplatin),Pegaspargase, Pemetrexed Disodium, Platinol-AQ (Cisplatin), Platinol(Cisplatin), Raloxifene Hydrochloride, Revlimid (Lenalidomide), Rituxan(Rituximab), Rituximab, Sclerosol Intrapleural Aerosol (Talc), SorafenibTosylate, Sprycel (Dasatinib), Sterile Talc Powder (Talc), Steritalc(Talc), Sunitinib Malate, Sutent (Sunitinib Malate), Synovir(Thalidomide), Talc, Tamoxifen Citrate, Tarabine PFS (Cytarabine),Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna(Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar(Temozolomide), Temozolomide, Temsirolimus, Thalomid (Thalidomide),Thalidomide, Totect (Dexrazoxane Hydrochloride), TopotecanHydrochloride, Torisel (Temsirolimus), Trastuzumab, Trisenox (ArsenicTrioxide), Tykerb (Lapatinib Ditosylate), Vectibix (Panitumumab),Velcade (Bortezomib), Vidaza (Azacitidine), Vorinostat, Xeloda(Capecitabine), Zinecard (Dexrazoxane Hydrochloride), Zoledronic Acid,Zolinza (Vorinostat) and Zometa (Zoledronic Acid).

According to a preferred embodiment of the invention the compound usefulin the treatment of bone mass decrease is a biphosphonate, a selectiveoestrogen receptor modulators (SERMs), a parathyroid hormone (PTH) (e.g.teriparatide (Forteo)), strontium ranelate, Denosumab or calcitonin, ora combination thereof. According to a more preferred embodiment, thebiphosphonate is chosen from the group comprising Alendronate (Fosamax,Fosamax Plus D), Etidronate (Didronel), Ibandronate (Boniva),Pamidronate (Aredia), Risedronate (Actonel, Actonel W/Calcium),Tiludronate (Skelid), and Zoledronic acid (Reclast, Zometa). Accordingto a more preferred embodiment, the SERMs is chosen from the groupcomprising raloxifene (Evista), bazedoxifene/premarin (Aprelal) andtamoxifen.

According to another embodiment, the present invention relates to theuse of the antibody, the nucleic acid sequence, the vector, thepharmaceutical composition or the kit of parts according to theinvention for the treatment of diseases associated to an increasedosteoclast activity. Such disease comprised but are not limited toendocrinopathies (hypercortisolism, hypogonadism, primary or secondaryhyperparathyroidism, hyperthyroidism), hypercalcemia, deficiency states(rickets/osteomalacia, scurvy, malnutrition), chronic diseases(malabsorption syndromes, chronic renal failure (renal osteodystrophy),chronic liver disease (hepatic osteodystrophy), drugs (glucocorticoids(glucocorticoid-induced osteoporosis), androgen deprivation therapy,aromatase inhibitor therapy, heparin, alcohol), and hereditary diseases(osteogenesis imperfecta, homocystinuria), osteoporosis, osteopetrosis,inflammation of bone associated with arthritis and rheumatoid arthritis,periodontal disease, fibrous dysplasia, and/or Paget's disease.

According to another embodiment, the present invention relates to theuse of the antibody, the nucleic acid sequence, the vector, thepharmaceutical composition or the kit of parts according to theinvention for the treatment of diseases associated to inflammationand/or autoimmunity. Such diseases comprise but are not limited toseronegative spondyloarthropathy (psoriatic arthritis, ankylosingspondylitis, reiters syndrome, spondyloarthropathy associated withinflammatory bowel disease), prosthetic joint loosening, connectivetissue diseases (juvenile rheumatoid arthritis, rheumatoid arthritis,systemic lupus erythematosus (SLE) and lupus nephritis, scleroderma,Sjogren's syndrome, mixed connective tissue disease, polymyositis,dermatomyositis), inflammatory bowel disease (e.g. Crohn's disease;ulcerative colitis), whipples disease, arthritis associated withgranulomatous ileocolitis, inflammatory skin conditions (autoimmunebullous pemphigoid, autoimmune pemphigus vulgaris, eczema, dermatitis),inflammatory lung disease (alveolitis, pulmonary fibrosis, sarcoidoisis,asthma, bronchitis, bronchiolitis obliterans), inflammatory renaldisease (glomerulonethritis, renal allograft rejection, renal tubularinflammation), atherosclerosis, systemic vasculitis (temporalarteritis/giant cell arteritis, takayasu arteritis, polyarteritisnodosa, Kawasaki disease, Wegener's granulomatosis, churg strausssyndrome, microscopic polyangiitis, necrotising glomerulonephritis,henoch schonlein purpura, essential cryoglobulinaemic vasculitis andother small vessel vasculitis, Behcets disease), macrophage activationdiseases (macrophage activation syndrome (MAS), adult onset stillsdisease, haemophagocytic syndrome), polymyalgia rheumatica, primarybiliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, Type 1Diabetes Mellitus, Hashimoto's thyroiditis, Graves' disease, multiplesclerosis (MS), Guillain-Barre syndrome, Addison's disease, and/orRaynaud's phenomenon, Goodpasture's syndrome.

According to another embodiment, the present invention relates to theuse of the antibody, the nucleic acid sequence, the vector, thepharmaceutical composition or the kit of parts according to theinvention for the treatment of cancer.

As used herein, the term “cancer” refers but is not limited toadenocarcinoma, acinic cell adenocarcinoma, adrenal cortical carcinomas,alveoli cell carcinoma, anaplastic carcinoma, basaloid carcinoma, basalcell carcinoma, bronchiolar carcinoma, bronchogenic carcinoma,renaladinol carcinoma, embryonal carcinoma, anometroid carcinoma,fibrolamolar liver cell carcinoma, follicular carcinomas, giant cellcarcinomas, hepatocellular carcinoma, intraepidermal carcinoma,intraepithelial carcinoma, leptomanigio carcinoma, medullary carcinoma,melanotic carcinoma, menigual carcinoma, mesometonephric carcinoma, oatcell carcinoma, squamal cell carcinoma, sweat gland carcinoma,transitional cell carcinoma, tubular cell carcinoma, amelioblasticsarcoma, angiolithic sarcoma, botryoid sarcoma, endometrial stromasarcoma, ewing sarcoma, fascicular sarcoma, giant cell sarcoma,granulositic sarcoma, immunoblastic sarcoma, juxaccordial osteogenicsarcoma, coppices sarcoma, leukocytic sarcoma (leukemia), lymphaticsarcoma (lympho sarcoma), medullary sarcoma, myeloid sarcoma(granulocitic sarcoma), austiogenci sarcoma, periosteal sarcoma,reticulum cell sarcoma (histiocytic lymphoma), round cell sarcoma,spindle cell sarcoma, synovial sarcoma, telangiectatic audiogenicsarcoma, Burkitt's lymphoma, NPDL, NML, NH and diffuse lymphomas.According to a preferred embodiment, the method according to theinvention is directed to the treatment of metastatic cancer to bone,wherein the metastatic cancer is breast, lung, renal, multiple myeloma,thyroid, prostate, adenocarcinoma, blood cell malignancies, includingleukemia and lymphoma; head and neck cancers; gastrointestinal cancers,including esophageal cancer, stomach cancer, colon cancer, intestinalcancer, colorectal cancer, rectal cancer, pancreatic cancer, livercancer, cancer of the bile duct or gall bladder; malignancies of thefemale genital tract, including ovarian carcinoma, uterine endometrialcancers, vaginal cancer, and cervical cancer; bladder cancer; braincancer, including neuroblastoma; sarcoma, osteosarcoma; and skin cancer,including malignant melanoma or squamous cell cancer.

The present invention further concerns a method for improving thetreatment of a cancer patient which is undergoing chemotherapeutictreatment with a cancer therapeutic agent, which comprises co-treatmentof said patient along with a method as above disclosed.

The present invention further concerns a method of improving cytotoxiceffectiveness of cytotoxic drugs or radiotherapy which comprisesco-treating a patient in need of such treatment along with a method asabove disclosed.

The present invention further concerns a method for improving thetreatment of a patient with a disease associated to an increasedosteoclast activity which is undergoing treatment with a biphosphonate,a selective oestrogen receptor modulators (SERMs), a parathyroid hormone(PTH) (e.g. teriparatide (Forteo)), strontium ranelate, Denosumab orcalcitonin, or a combination thereof, which comprises co-treatment ofsaid patient along with a method as above disclosed.

In another embodiment use of an antibody of the invention iscontemplated in the manufacture of a medicament for preventing ortreating metastatic cancer to bone in a patient suffering frommetastatic cancer. In a related embodiment, the metastatic cancer isbreast, lung, renal, multiple myeloma, thyroid, prostate,adenocarcinoma, blood cell malignancies, including leukemia or lymphoma;head or neck cancers; gastrointestinal cancers, including esophagealcancer, stomach cancer, colon cancer, intestinal cancer, colorectalcancer, rectal cancer, pancreatic cancer, liver cancer, cancer of thebile duct or gall bladder; malignancies of the female genital tract,including ovarian carcinoma, uterine endometrial cancers, vaginalcancer, or cervical cancer; bladder cancer; brain cancer, includingneuroblastoma; sarcoma, osteosarcoma; or skin cancer, includingmalignant melanoma or squamous cell cancer.

According to another embodiment, the present invention relates to theuse of the antibody, the nucleic acid sequence, the vector, thepharmaceutical composition or the kit of parts according to theinvention in the manufacture of a medicament.

According to another embodiment, the present invention relates to theuse of the antibody, the nucleic acid sequence, the vector, thepharmaceutical composition or the kit of parts according to theinvention in the manufacture of a medicament for treating a patienthaving cancer.

According to another embodiment, the present invention relates to theuse of the antibody, the nucleic acid sequence, the vector, thepharmaceutical composition or the kit of parts according to theinvention in the manufacture of a medicament for treating a patienthaving a disease associated to an increased osteoclast activity.

According to another embodiment, the present invention relates to theuse of the antibody, the nucleic acid sequence, the vector, thepharmaceutical composition or the kit of parts according to theinvention in the manufacture of a medicament for treating a patienthaving an inflammatory disease, more specifically an inflammatory boweldisease.

According to another embodiment, the present invention relates to theuse of the antibody, the nucleic acid sequence, the vector, thepharmaceutical composition or the kit of parts according to theinvention in the manufacture of a medicament for treating a patientsuffering from rheumatoid arthritis.

Administering the antibody, the nucleic acid sequence, the vector, thepharmaceutical composition or the kit of parts according to theinvention may be accomplished by any means known to the skilled artisan.Preferred routes of administration include but are not limited tointradermal, subcutaneous, oral, parenteral, intramuscular, intranasal,sublingual, intratracheal, inhalation, ocular, vaginal, and rectal.According to a preferred embodiment the antibody, the nucleic acidsequence, the vector, the pharmaceutical composition or the kit of partsaccording to the invention are delivered systemically.

The administration may take place in a single dose or a dose repeatedone or several times after a certain time interval. Desirably, theantibody, the nucleic acid sequence, the vector, the pharmaceuticalcomposition or the kit of parts according to the invention areadministered 1 to 10 times at weekly intervals.

For general guidance, suitable dosage for the antibody is about 2 mg/kgto 30 mg/kg, 0.1 mg/kg to 30 mg/kg or 0.1 mg/kg to 10 mg/kg body weight.Suitable dosage for the vector according to the invention varies fromabout 10⁴ to 10¹⁰ pfu (plaque forming units), desirably from about 10⁵and 10⁸ pfu for MVA vector whereas it varies from about 10⁵ to 10¹³ iu(infectious units), desirably from about 10⁷ and 10¹² iu for adenovirusbased vector. A composition based on vector plasmids may be administeredin doses of between 10 μg and 20 mg, advantageously between 100 μg and 2mg.

When the use or the method according to the invention is for thetreatment of cancer, the method or use of the invention can be carriedout in conjunction with one or more conventional therapeutic modalities(e.g. radiation, chemotherapy and/or surgery). The use of multipletherapeutic approaches provides the patient with a broader basedintervention. In one embodiment, the method of the invention can bepreceded or followed by a surgical intervention. In another embodiment,it can be preceded or followed by radiotherapy (e.g. gamma radiation).Those skilled in the art can readily formulate appropriate radiationtherapy protocols and parameters which can be used (see for examplePEREZ. Principles and practice of radiation oncology. 2nd edition.LIPPINCOTT, 1992.)

BRIEF DESCRIPTION OF FIGURES IN THE DRAWINGS

FIG. 1 depicts the specific staining of CSF-1R-transfected NIH/3T3 cellsby mAb CXIIG6.

FIG. 2 shows the inhibition of CSF-1 binding to cell-surface CSF-1R inpresence of mAb CXIIG6.

FIG. 3 shows the specific blockade of soluble human CSF-1R by mAb CXIIG6(‘ctrl’ means control; ‘neg SN’ means negative control hybridomasupernatant).

FIG. 4 shows the inhibition of human osteoclast differentiation andsecretion of matrix-metalloprotease-9 (MMP-9) in presence of mAb CXIIG6(‘ctrl’ means control; ‘CXIIG6 SN’ means CXIIG6 hydridoma supernatant;‘neg SN’ means negative control hybridoma supernatant).

FIG. 5 shows the non-cross-reactivity of mAb CXIIG6 with other tyrosinekinase receptors having homology to CSF-1R (‘SN’ means hydridomasupernatant).

FIG. 6 shows the nucleic acid sequence (SEQ ID NO: 1) and deduced aminoacid sequence (SEQ ID NO: 2) of the CXIIG6 heavy chain. The primersequences including restriction sites for cloning added to thenucleotide sequences are underlined. The restriction sites are shown inunderlined italic type. The amino acid sequences of the V-domains arehighlighted in bold type.

FIG. 7 shows the nucleic acid sequence (SEQ ID NO: 3) and deduced aminoacid sequence (SEQ ID NO: 4) of the CXIIG6 light chain. The primersequences including restriction sites for cloning added to thenucleotide sequences are underlined. The restriction sites are shown inunderlined italic type. The amino acid sequences of the V-domains arehighlighted in bold type.

FIG. 8 shows the plasmid construct pTG17753.

FIG. 9 shows the plasmid construct pTG17727.

FIG. 10 shows the plasmid construct pOptiVEC™.

FIG. 11 shows the plasmid construct pTG17895.

FIG. 12 shows the plasmid construct pTG17812.

FIG. 13 shows the plasmid construct pTG17868.

FIG. 14 shows the plasmid construct pTG17869.

FIG. 15 shows humanized CXIIG6 light chain variants.

FIG. 16 shows humanized CXIIG6 IgG1 heavy chain variants.

FIG. 17 shows the specific blockade of soluble human CSF-1R byrecombinant murine CXIIG6 and chimeric CXIIG6 IgG1.

FIG. 18 shows, the inhibition of human osteoclast differentiation andsecretion of matrix-metalloprotease-9 (MMP-9) in presence of recombinantmurine CXIIG6 and chimeric CXIIG6 IgG1.

MODE(S) FOR CARRYING OUT THE INVENTION Specific Staining ofCSF-1R-Transfected NIH/3T3 Cells by mAb CXIIG6

The B4-800-5 cell line was generated by stable transfection of NIH/3T3cells with an expression plasmid encoding the full-length human CSF-1R.Cell-surface CSF-1R expression on B4-800-5 cells was verified byindirect immunostaining with the anti-human CSF-1R mAbs 61701 (mouseIgG₁, R&D Systems) or 2-4A5-4 (rat IgG_(1,k), GeneTex), compared toisotype controls (FIG. 1, upper and middle panels). Culture supernatantsfrom hybridoma CXIIG6 or from a negative control hybridoma were used forimmunostaining B4-800-5 cells or parental NIH/3T3 cells (FIG. 1, lowerpanels).

Flow cytometry analysis showed that culture supernatant from hybridomaCXIIG6 selectively stained B4-800-5 cells, demonstrating the mAbspecificity for cell-surface CSF-1R.

Inhibition of CSF-1 Binding to Cell-Surface CSF-1R

3×10⁵ THP-1 cells (human CSF-1R-positive monocytic leukemia cell line)were incubated for 30 min at 4° C. in the presence of either hybridomaculture supernatants, serum from a naive or an anti-CSF-1R-immunizedmouse (dilution 1:1000), mAb anti-CSF-1R 2-4A5-4 (GeneTex) or a controlrat IgG₁ (10 μg/ml), or no reagent. After two washes with cold PBS,cells were incubated with 1 μg/ml biotinylated recombinant human CSF-1for 30 min. Cells were washed twice and further incubated for 30 min at4° C. with 10 μg/ml streptavidin-Alexa Fluor 488 (Invitrogen). Afterwashing with PBS and fixation with 4% paraformaldehyde, cell stainingwas analyzed by flow cytometry.

Decreased fluorescence intensities compared to control samples reflectthe inhibition of CSF-1 binding to cell-surface CSF-1R. Serum from aCSF-1R-immunized mouse blocks CSF-1 binding to THP-1 cells (FIG. 2).While negative control hybridoma supernatant or an irrelevant mAb showno effect, culture supernatant from hybridoma CXIIG6 inhibits CSF-1binding to THP-1 cells (FIG. 2, lower right panel), as does mAb 2-4A5-4(lower left panel).

Localization of mAb CXIIG6 Binding Site

To identify the binding site of mAb CXIIG6 on the CSF-1R, a Western blotwas performed using soluble forms of the human CSF-1R comprising eitherthe five extracellular immunoglobulin-like domains (Met 1 to Glu 512,R&D Systems) or only the three N-terminal immunoglobulin-like domains ofthe extracellular region of CSF-1R (Met 1 to Ser 290), both fused attheir C-terminal ends to the Fc region of a human IgG₁. A soluble formof the EGFR fused to human IgG₁ Fc (R&D Systems) was used as a negativecontrol.

Hundred nanograms of each soluble receptor were submitted toelectrophoresis in native conditions before transfer to nitrocellulosesheet and probing with either hybridoma supernatants, rabbit pAbc-fms/CSF-1R H300 (Santa Cruz Biotechnology), mouse mAb 61701 (R&DSystems) or serum from naive or CSF-1R-immunized mice.

Both soluble forms of the CSF-1R were detected as broad bands whenprObed with pAb c-fms/CSF-1R H300, mAb 61701 or serum from the immunizedmouse. No detectable signals were observed with naive mouse serum or anegative control hybridoma supernatant. CXIIG6 hybridoma supernatantrecognized CSF-1R₁₋₂₉₀:Fc as well as CSF-1R₁₋₅₁₂:Fc, but not EGFR:Fc,indicating that CXIIG6 binds specifically to an epitope lying within thethree N-terminal immunoglobulin-like domains (between residues 1 to 290)of the human CSF-1R.

Specific Blockade of Soluble Human CSF-1R by mAb CXIIG6

The CSF-1-dependent murine myeloid leukemia M-NFS-60 cell line(#CRL-1838, ATCC) was used to assess the blocking activity of CXIIG6hybridoma supernatant on human and murine CSF-1R. Five nanograms ofsoluble human CSF-1R (CSF-1R₁₋₅₁₂:Fc from R&D Systems) were preincubatedin white 96-well microplates with serial dilutions of either hybridomasupernatants, mAb 61701 (R&D Systems) or murine isotype control mAb.10E4 M-NFS-60 cells cultured overnight in the absence of CSF-1 were thenadded into the culture wells together with 0.1 ng of human CSF-1 in afinal assay volume of 100 μl. Cultures were incubated for 48 h at 37° C.and proliferation was quantified by BrdU incorporation using a CellProliferation ELISA (Roche).

Soluble human CSF-1R completely inhibited the proliferation of M-NFS-60cells mediated by human CSF-1, as shown in the presence of negativehybridoma supernatant containing or not a negative control IgG₁ (FIG. 3;mean+/−SEM of 3 wells). In contrast, CXIIG6 hybridoma supernatant andpositive control mAb 61701 were both able to restore cell proliferationin a dose-dependent manner, showing that they were able to neutralizesoluble human CSF-1R.

In this assay, active M-NFS-60 cell proliferation in the presence of lowdilutions of CXIIG6 hybridoma supernatant showed that mAb CXIIG6 wasunable to block the murine CSF-1R expressed by M-NFS-60 cells. Moreover,in a murine CSF-1-supported M-NFS-60 proliferation assay performed inthe absence of soluble CSF-1R, treatment with mAb AFS98 anti-mouseCSF-1R (eBioscience) resulted in a dramatic concentration-dependentdecrease of cell growth (data not shown). CXIIG6 hybridoma supernatant,like negative control antibodies and negative hybridoma supernatant,caused no reduction in cell proliferation. These results demonstratethat mAb CXIIG6 specifically targets human CSF-1R.

Inhibition of Human Osteoclast Differentiation and Secretion ofMatrix-Metalloprotease-9 (MMP-9)

Osteoclasts, were generated from human monocytes obtained by elutriationof PBMCs from a healthy blood donor. In brief, monocytes were seeded at2×10E4 cells per well in 96-well plates and treated for 45 min witheither hybridoma culture supernatants, mAbs anti-human CSF-1R 61701 (R&DSystems) or 2-4A5-4 (GeneTex), mAb anti-human CSF-1 26730 (R&D Systems),murine or rat isotype controls, or sera from naive and CSF-1R-immunizedmice diluted in hybridoma culture medium. Complete a-MEM medium wasadded to the culture wells with or without human CSF-1 and RANKL(PeproTech, 25 and 40 ng/ml respectively). Hybridoma supernatants, mAbsor/and medium with or without cytokines were replenished every 3 daysfor 9 days. Conditioned culture supernatants were harvested on day 9 andassayed for total human MMP-9 using an ELISA assay (R&D Systems).Osteoclast formation was evaluated by staining of tartrate-resistantacid phosphatase (TRAP) using the leukocyte acid phosphatase kit fromSigma-Aldrich.

CSF-1+RANKL induced monocytes to differentiate into osteoclasts, definedas large multinucleated TRAP-positive cells, whereas no TRAP-positiveosteoclasts were obtained in the absence of cytokines. Addition of 0.5μg/ml anti-CSF-1 mAb 26730 completely abrogated osteoclastdifferentiation, as shown by lack of MMP-9 secretion. Anti-CSF-1R mAbs61701 or 2-4A5-4 at the same concentration and immunized mouse serum(dilution 1:1000) inhibited osteoclast formation only partially (FIG. 4;with (+) or without (−) cytokines; mean+/−SEM of 3 wells; *: mean of 2wells). Treatment with CXIIG6 hybridoma culture supernatant diluted 1:20or 1:100 significantly reduced the level of MMP-9 production, comparedwith two negative control hybridoma supernatants (A, B). These resultsdemonstrate that mAb CXIIG6 inhibits the differentiation of osteoclastsfrom human monocytes by blocking the function of cell surface CSF-1R.

Inhibition of the CSF-1-Dependent Phosphorylation of CSF-1R

The B4-800-5 cell line obtained by stable transfection of NIH/3T3 cellswith a plasmid expressing human CSF-1R was used to investigate theeffect of CXIIG6 hybridoma supernatant on CSF-1-dependent CSF-1Rphosphorylation. Cells were seeded at 2×10E5 cells per 60-mm Petri dishand cultured for 48 to 72 h. Following serum deprivation for 1 h at 37°C., cells were treated for 1 h at 37° C. with culture medium containingeither CXIIG6 hybridoma supernatant, mAb 2-4A5-4 (NeoMarkers) or isotypecontrol mAbs (diluted in negative hybridoma supernatant), and thenstimulated with 100 ng/ml hCSF-1 or left unstimulated for 5 min at 37°C. Cell layers were then lysed and total proteins were extracted. Ten μgproteins were analyzed by probing Western blots with either the rabbitpAb c-fms/CSF-1R H300 or the rabbit pAb p-c-fms/CSF-1R (Tyr708)-R (SantaCruz Biotechnology), followed by goat anti-rabbit immunoglobulinsHRP.

In the absence of CSF-1, neither CXIIG6 hybridoma supernatant nor mAb2-4A5-4 induced receptor phosphorylation as seen with the antibodyspecific for CSF-1R phosphorylated at position 708, showing that mAbCXIIG6 alone does not exert an agonistic effect. Upon stimulation withCSF-1, the amount of CSF-1R decreased in isotype control-treated cellscompared with unstimulated cells, and CSF-1R was phosphorylated onTyr708 (data not shown). Pretreatment with CXIIG6 hybridoma supernatantor with mAb 2-4A5-4 did not enhance CSF-1R disappearance.Phosphorylation of CSF-1R was decreased following treatment with CXIIG6hybridoma supernatant or with mAb 2-4A5-4. These results show that mAbCXIIG6 is able to block the CSF-1-dependent phosphorylation of CSF-1R.

Cross-Reactivity of mAb CXIIG6

The cross-reactivity of mAb CXIIG6 was tested by ELISA on a series ofpurified soluble receptors belonging to the type III subfamily oftyrosine kinase receptors and showing homology to CSF-1R in theirextracellular Ig-like domains: soluble VEGFR-1, VEGFR-2, Flt-3 andPDGFRβ (all four expressed as Fc fusion proteins), as well as PDGFRα andSCFR (c-kit) were obtained from R&D Systems and used for coating anELISA plate. Soluble EGFR(R&D Systems), from the EGFR subfamily oftyrosine kinase receptors, was used as a negative control.

Culture supernatants from either hybridoma CXIIG6 (CXIIG6 SN) or anegative control hybridoma, or the anti-CSF-1R mouse IgG₁ 61701 (R&DSystems) were incubated on the coated ELISA plate at antibodyconcentrations of 500 ng/ml. After washing the ELISA plate, boundantibodies were revealed using peroxidase-conjugated goat anti-mouse Ig(Sigma) and OD_((450-540 nm)) was measured. Results depicted in FIG. 5show that like mAb 61701, CXIIG6 strongly bound the CSF-1R while nospecific signal was detected on any other tyrosine kinase receptor. Thisshows that among the various type III tyrosine kinase receptors tested,CXIIG6 is specific for CSF-1R.

Construction of Expression Vectors for mAb CXIIG6

The OptiCHO™ Antibody Express Kit (Invitrogen, Catalog No. 12762-019)was used for the cloning of the genes encoding the CXIIG6 heavy andlight chains in order to produce the mAb CXIIG6 in DG44 mammalian cellline. The OptiCHO™ Antibody Express Kit includes: (1) The pOptiVEC™vector, a bicistronic plasmid that allows the cloning of the gene ofinterest downstream of CMV promoter. The transcription of the gene ofinterest is separated from the dihydrofolate reductase (DHFR)auxotrophic selection marker by an internal ribosome entry site (IRES),allowing transcription of the gene of interest and of the selectionmarker on the same mRNA; (2) The pcDNA™ 3.3 vector that allows thecloning of the gene of interest downstream of CMV promoter. The pcDNA™3.3 contains a neomycin resistance gene allowing selection usingGeneticin®. The pOptiVEC™ and pcDNA™ 3.3 vectors contain the TK poly-Asequence which directs proper processing of the 3′ end of the mRNA ofthe gene of interest.

Specific primers (see Table 4) were synthesized and used for the PCRamplification and cloning of the entire CXIIG6 heavy chain and lightchain genes (respectively SEQ ID NO:1 and SEQ ID NO:3; see respectivelyFIG. 6 and FIG. 7). The backward primers included the Kozak consensussequence for efficient eukaryotic translation (KOZAK M. An analysis of5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic AcidsRes. 1987, 15(20): 8125-8148.).

TABLE 4 Primer Sequence OTG18929 GCCGCCACCATGTACTTGGGACTGAACTATGTATTC(SEQ ID NO: 30) OTG18930 GGAGATCTTCATTTACCCGGAGTCCGGGA (SEQ ID NO: 31)OTG18931 GCCGCCACCATGAGTGTGCCCACTCAGGTCCTG (SEQ ID NO: 32) OTG18932GCCCGGGCTAACACTCATTCCTGTTGAAGCTC (SEQ ID NO: 33)

CXIIG6 heavy chain was PCR amplified by using OTG18929 and OTG18930 withplasmid pTG17753 (FIG. 8) as template, and cloned into the vectorpOptiVEC™-TOPO® (pOptiVEC™-TOPO® TA Cloning® Kit, Invitrogen, Catalogno. 12744-017-01) and the pcDNA™ 3.3-TOPO® vectors (pcDNA™ 3.3-TOPO® TACloning® Kit, Invitrogen, Catalog No. K8300-01) to obtain respectivelypTG17786 and pTG17789.

CXIIG6 light chain was PCR amplified by using OTG18931 and OTG18932 withplasmid pTG17727 (FIG. 9) as template, and cloned into the vectorpOptiVEC™-TOPO® (pOptiVEC™-TOPO® TA Cloning® Kit, Invitrogen, Catalogno. 12744-017-01) and the pcDNA™ 3.3-TOPO® vectors (pcDNA™ 3.3-TOPO® TACloning® Kit, Invitrogen, Catalog No. K8300-01) to obtain respectivelypTG17788 and pTG17787.

The nucleotide sequence of the whole expression cassette, including CMVpromoter and TK polyA signal of pTG17786, pTG17787, pTG17788 andpTG17789 were sequenced and found in compliance with their theoreticalsequences.

Generation of Chimeric Antibodies from mAb CXIIG6

The variable domains of mAb CXIIG6 were combined with human constantregions.

To generate the chimeric light chain (named Chimeric CXIIG6 Iv chain), atheorical sequence was designed by joining the sequence encoding theCXIIG6 VK Domain (from SEQ ID NO:3) to the sequence encoding for thehuman IGKC region (GenBank accession number: J00241). This XbaI NotI DNAfragment kept the same non translated sequence at 5′ end, includingKozak sequence, as the one used in the murine version (as in pTG17787and pTG17788 described above). The chimeric CXIIG6 light chain sequencewas codon optimized for expression in CHO, assembled from syntheticoligonucleotides, and subcloned into pOptiVEC™ (FIG. 10) via XbaI NotIby GeneArt AG. The obtained chimeric CXIIG6 light chain (variable andconstant regions) codon optimized nucleic acid sequence is as set forthin SEQ ID NO:34. The obtained plasmid was named pTG17895 (FIG. 11).

To generate the chimeric heavy IgG1 and IgG4 chains (respectively namedChimeric CXIIG6 IgG1 and Chimeric CXIIG6 IgG4 chains), the theoricalsequences were designed by joining the sequence encoding the CXIIG6 VHDomain (from SEQ ID NO:1) to the sequences encoding either for the humanIGHG1C region (GenBank accession number: J00228) or for the human IGHG4Cregion (GenBank accession number: K01316). These XbaI NotI DNA fragmentskept the same non translated sequence at 5′ end, including Kozaksequence, as the one used in the murine version (as in pTG17786 andpTG17789 as described above). The chimeric CXIIG6 heavy chains were thencodon optimized for expression in CHO, synthesized and cloned intopTG17812 (FIG. 12) via XbaI NotI by GeneArt AG. The obtained chimericCXIIG6 IgG1 heavy chain (variable and constant regions) codon optimizednucleic acid sequence is as set forth in SEQ ID NO:35 and the obtainedplasmids was named pTG17868 (FIG. 13). The obtained chimeric CXIIG6 IgG4heavy chain (variable and constant regions) codon optimized nucleic acidsequence is as set forth in SEQ ID NO:36 and the obtained plasmids wasnamed pTG17869 (FIG. 14).

Generation of Human Antibodies from mAb CXIIG6

To generate the humanized light chain variants, amino acid substitutionsaccording to Table 2 were performed within the light-chain variableregion as set forth in SEQ ID NO:9.

DNA sequences were designed by joining modified sequences bearingsubstitutions of the CXIIG6 VK Domain (from SEQ ID NO:3) to the sequenceencoding for the human IGKC region (GenBank accession number: J00241).This XbaI NotI DNA fragment kept the same non translated sequence at 5′end including Kozak sequence as the one used in the murine version (asin pTG17787 and pTG17788 as described above). The humanized CXIIG6 lightchain sequences were then codon optimized for expression in CHO,assembled from synthetic oligonucleotides and cloned into pOptiVEC™(FIG. 10) via XbaI NotI by GeneArt AG. The obtained humanized CXIIG6light chain variants and plasmids are listed in FIG. 15.

To generate the humanized heavy chain variants, amino acid substitutionsaccording to Table 1 were performed within the heavy-chain variableregion as set forth in SEQ ID NO:6.

DNA sequences were designed by joining modified sequences bearingsubstitutions of the CXIIG6 VH Domain (from SEQ ID NO:1) to thesequences encoding for the human IGHG1C region (GenBank accessionnumber: J00228). The XbaI ApaI DNA fragments kept the same nontranslated sequence at 5′ end including Kozak sequence as used the onein the murine version (as in pTG17786 and pTG17789 as described above).The DNA sequences were then codon optimized for expression in CHO,synthesized and cloned into pTG17812 (FIG. 12) via XbaI ApaI by GeneArtAG. The obtained humanized CXIIG6 IgG1 heavy chain variants and plasmidsare listed in FIG. 16.

In Vitro Inhibitory Activities of Recombinant Murine CXIIG6 and ChimericCXIIG6 IgG1

To determine whether purified recombinant murine CXIIG6 (as previouslydescribed) and its chimeric IgG1 variant (chimeric CXIIG6 IgG1 aspreviously described) were able to block soluble human CSF-1R,dose-response studies were performed in the M-NFS-60 cell proliferationand osteoclast differentiation models (as previously described).Purified polyclonal murine IgG2a from Rockland (Rockland, 010-0141) anda chimeric IgG1 produced by the applicant were tested in parallel ascontrol antibodies. Blocking effect was evaluated by exposing cells toconcentration ranges of active anti-CSF-1R antibodies, as measured byantigen binding in a SPR biosensor assay. Comparison between mAbs CXIIG6and their respective control mAb was done by loading equal amounts oftotal antibody (SPR biosensor assay by Fc binding).

M-NFS-60 bioassay: In the M-NFS-60 bioassay, cells were treated with0.23 ng/ml to 0.5 μg/ml active mAbs CXIIG6 (recombinant murine CXIIG6;chimeric CXIIG6 IgG1) or corresponding concentrations of control mAbs inthe presence of 50 ng/ml human soluble CSF-1R and 1 ng/ml human CSF-1for 48 h. Results depicted in FIG. 17 show that M-NFS-60 cell growthincreased in response to increasing concentrations of both mAbs CXIIG6(recombinant murine CXIIG6; chimeric CXIIG6 IgG1) demonstrating thatthey antagonized the binding of soluble CSF-1R to CSF-1 (mean+/−SEM oftriplicate wells). Chimeric CXIIG6 IgG1 was as effective as recombinantmurine CXIIG6 in restoring cell proliferation. Control murine IgG2a andchimeric IgG1 had no effect on CSF-1 neutralization by soluble CSF-1Rover their respective concentration range.

Osteoclast bioassay: In the osteoclast bioassay, elutriated humanmonocytes were incubated for 8 days with 0.85 ng/ml to 0.62 μg/ml activemAbs CXIIG6 (recombinant murine CXIIG6; chimeric CXIIG6 IgG1) in thepresence of 25 ng/ml CSF-1 (ImmunoTools) and 40 ng/ml RANKL. The mediumand all added agents were replenished on day 4 and 6 and total MMP-9 wasmeasured in culture media conditioned from day 6 to 8. Results depictedin FIG. 18 show that in comparison with control antibodies, recombinantmurine CXIIG6 and its chimeric variant (chimeric CXIIG6 IgG1) eachsignificantly reduced MMP-9 production which parallels osteoclastdifferentiation, indicating that growth retardation occurred (FIG. 18;mean+/−SEM of triplicate wells).

Taken together, these results further demonstrate that purifiedrecombinant murine CXIIG6 and chimeric CXIIG6 IgG1 inhibit cell-surfaceas well as soluble human CSF-1R.

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1.-66. (canceled)
 67. An isolated nucleic acid sequence encoding anantibody that specifically binds to human CSF-1R, the antibodycomprising a heavy chain variable region comprising (a) a CDR1comprising the amino acid sequence starting at position 45 and finishingat position 54 of SEQ ID NO:2, (b) a CDR2 comprising the amino acidsequence starting at position 66 and finishing at position 87 of SEQ IDNO:2, and (c) a CDR3 comprising the amino acid sequence starting atposition 117 and finishing at position 126 of SEQ ID NO:2; and a lightchain variable region comprising (a) a CDR1 comprising the amino acidsequence starting at position 44 and finishing at position 56 of SEQ IDNO:4, (b) a CDR2 comprising the amino acid sequence starting at position66 and finishing at position 76 of SEQ ID NO:4, and (c) a CDR3comprising the amino acid sequence starting at position 109 andfinishing at position 117 of SEQ ID NO:4.
 68. The nucleic acid sequenceof claim 67, wherein the heavy chain variable region of the antibodycomprises the amino acid sequence as set forth in SEQ ID NO:6.
 69. Thenucleic acid sequence of claim 68, wherein the heavy chain variableregion of the antibody consists of SEQ ID NO:6.
 70. The nucleic acidsequence of claim 67, wherein the light chain variable region of theantibody comprises the amino acid sequence as set forth in SEQ ID NO:9.71. The nucleic acid sequence of claim 70, wherein the light chainvariable region of the antibody consists of SEQ ID NO:9.
 72. The nucleicacid sequence of claim 68, wherein the light chain variable region ofthe antibody comprises the amino acid sequence as set forth in SEQ IDNO:9.
 73. The nucleic acid sequence of claim 68, wherein the encodedantibody is a humanized antibody.
 74. The nucleic acid sequence of claim73, wherein the light chain variable region of the antibody comprisesthe amino acid sequence as set forth in SEQ ID NO:9.
 75. The nucleicacid sequence of claim 67, wherein the heavy chain variable region ofthe antibody comprises the amino acid sequence as set forth in SEQ IDNO:6 modified by the amino acid substitutions K3Q, E5V, M18L, and P90Aand wherein the light chain variable region of the antibody comprisesthe amino acid sequence as set forth in SEQ ID NO:9.
 76. The nucleicacid sequence of claim 67, wherein the heavy chain variable region ofthe antibody comprises the amino acid sequence as set forth in SEQ IDNO: 6 modified by the amino acid substitutions K3Q, E5V, M18L, K19R,540A, E42G, M43K R89K, P90T, 195V, T113L, and L114V, and wherein thelight chain variable region of the antibody comprises the amino acidsequence as set forth in SEQ ID NO:9.
 77. An isolated nucleic acidsequence coding for an antibody that specifically binds to human CSF-1R,the antibody comprising: a heavy chain variable region comprising theamino acid sequence as set forth in SEQ ID NO:6, and a light chainvariable region comprising the amino acid sequence as set forth in SEQID NO:9 modified by the amino acid substitutions A9S, E17D, T18R, Q40P,S43A, Q45K, A55E, D56S, Q70D, S72T, K74T, N76S, S80P, S85T, and G100Q.78. An isolated nucleic acid sequence coding for an antibody thatspecifically binds to human CSF-1R, the antibody comprising: a heavychain variable region comprising the amino acid sequence as set forth inSEQ ID NO:6, and a light chain variable region comprising the amino acidsequence as set forth in SEQ ID NO:9 modified by the amino acidsubstitutions A9S, V13A, E17D, T18R, Q40D, K74T, N76S, and 580P.
 79. Anucleic acid sequence coding for an isolated antibody that specificallybinds to human CSF-1R, comprising: a heavy chain variable regioncomprising the amino acid sequence as set forth in SEQ ID NO:6, and alight chain variable region comprising the amino acid sequence as setforth in SEQ ID NO:9 modified by the amino acid substitutions A9S, V13A,E17D, T18R, Q40P, S43A, Q45K, V48L, D56S, Q70D, S72T, K74T, N76S, 580P,G84A, S85T, and G100Q.
 80. The nucleic acid sequence of claim 67,wherein the antibody is a polyclonal antibody, monoclonal antibody, Fab,F (ab′)2, Fv, scFv, antigen binding fragment, or diabody.
 81. Thenucleic acid sequence of claim 80, wherein the antibody is a monoclonalantibody.
 82. The nucleic acid sequence of claim 67, wherein theantibody has a binding affinity for human CSF-1R of at least 10⁹ M⁻¹.83. A vector comprising the nucleic acid sequence of claim
 67. 84. A kitcomprising the vector of claim
 83. 85. A composition comprising thevector of claim 83 and a pharmaceutically acceptable carrier.